Multi-well microfiltration apparatus and method for avoiding cross-contamination

ABSTRACT

The present invention provides multi-well plates and column arrays in which samples (e.g., cell lysates containing nucleic acids of interest, such as RNA) can be analyzed and/or processed. In one embodiment, the microfiltration arrangement is a multilayer structure, including (i) a column plate having an array of minicolumns into which samples can be placed, (ii) a discrete filter element disposed in each minicolumn, (iii) a drip-director plate having a corresponding array of drip directors through which filtrate may egress, and (iv) a receiving-well plate having a corresponding array of receiving wells into which filtrate can flow. The invention provides multi-well microfiltration arrangements that are relatively simple to manufacture and that overcome many of the problems associated with the prior arrangements relating to (i) cross-contamination due to wicking across a common filter sheet or (ii) individual filter elements entrapping sample constituents within substantial dead volumes. Further, the invention provides multi-well microfiltration arrangements that adequately support discrete filter elements disposed in the wells without creating substantial preferential flow. Additionally, the invention provides multi-well microfiltration arrangements that avoid cross-contamination due to aerosol formation, pendent drops and/or splattering. Other disclosed features of the invention provide for the automated covering or heat-sealing of filtrate samples separately collected in an array of wells.

This application is a division of Ser. No. 09/182,946, filed on Oct. 29,1998, U.S. Pat. No. 6,159,368, which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to multi-well plates and column arrays inwhich samples are analyzed or processed.

BACKGROUND OF THE INVENTION

In recent years, microtitration wells have assumed an important role inmany biological and biochemical applications, such as samplepreparation, genome sequencing, and drug discovery programs. A varietyof multi-well arrangements, constructed according to standardizedformats, are now popular. For example, a tray or plate having ninety-sixdepressions or cylindrical wells arranged in a 12×8 regular rectangulararray is one particularly popular arrangement.

In some multi-well constructions, a filter sheet or membrane is heldagainst the lower ends, or lips, of open-bottomed wells. Such plates areoften manufactured as a multi-layered structure including a unitarysheet of filter material disposed to cover the bottom apertures of allthe wells, the filtration sheet being sealed to the outer lip of one ormore of the well apertures. The use of a single sheet of filter materialin such a manner, however, can lead to cross-contamination betweenadjacent wells due to the ability of liquid to disperse, e.g., bywicking, across the sheet.

In an effort to overcome this problem, it has been proposed to provideeach well with its own discrete filter element or disc. According to onesuch design, a pre-cut filter disc is inserted into an upper, open endof each well and pushed down until it rests at the bottom of the well.An O-ring is then press-fit down into each well until it comes to restagainst the top of the filter disc. The O-ring frictionally engages thecolumn inner wall, thereby retaining the filter in place. While avoidingthe cross-contamination problems of unitary filter sheets, such aconstruction is obviously cumbersome to manufacture. Also, the portionof the disk that gets pinched between the O-ring and the floor of thewell introduces a significant “dead volume,” which can have an adverseimpact on sample purification. For example, sample matrix can becomeentrapped in these areas along a significant portion of the peripheraledge of individual filter discs. When purifying DNA from blood samples,entrapment of small amounts of hemoglobin (heme) on the edges of acellulose blot membrane will eventually contaminate the final product inthe last stages of the purification process. The contaminating hemeresidue is a strong inhibitor in PCR and sequencing reaction assays ofthe DNA products.

Another multi-well arrangement, wherein each well has its own discretefilter element, is formed by positioning a single sheet of filtermaterial between an upper plate, having a plurality of mini-columnsformed therein, and a lower plate having a plurality of corresponding“drip directors.” Upon bringing the plates together and forming anultrasonic bond therebetween, the filter sheet is die-cut intoindividual filter discs positioned below respective mini-columns.Although this construction is easier to manufacture than the abovearrangement, it suffers similar disadvantages. Specifically, asubstantial portion of each filter disc's peripheral edge becomespinched between the column plate and the drip director plate, resultingin a significant dead volume that can adversely impact samplepurification.

There is, thus, a need for a multi-well microfiltration arrangement thatis relatively simple to manufacture, and that overcomes the problemsassociated with the prior arrangements relating to cross-contaminationdue to wicking across a common filter sheet, or individual filter discsentrapping sample constituents within substantial dead volumes.

Most of the known multi-well filtration plates, and particularly thoseproviding an individual filter disc for each well, lack adequate spacebelow the filter element to permit an evenly distributed flow of fluidacross the filter. In many arrangements, a drip director, at the bottomof each well, provides an expansive, flat surface upon which much of thefilter element rests. Preferential flow pathways are thereby created,favoring those areas of the filter element that are not in contact with,or in close proximity to, the drip director surface. Such preferentialflow can have an adverse impact on the elution of solutes. For example,preferential flow pathways can impede the leaching of retained sampleconstituents in non-favored regions of the filter element.

On the other hand, a lack of adequate support beneath each filterelement can be problematic, as well. The filter media used in multi-welltrays are typically quite thin and exhibit relatively poor mechanicalproperties. In certain stressful situations, e.g., high-pressure orvacuum filtration, such membranes may not maintain their integrity.Filter discs that are supported only about their peripheral edges mightsag, particularly along their central regions, and may even pull loosefrom the structure holding their edges. For example, a filter disc mightcollapse into the cavity of a drip director. This would affect theporosity of the filter, trapping certain sample constituents in thefilter that would otherwise elute. Moreover, if a bypass forms along theedges of the filter, due to the filter disc pulling away from theperipheral supporting structure, an undesirable loss of sample mayresult.

There is, thus, a need for a multi-well microfiltration arrangement thatadequately supports the filter media at each well, without creatingsubstantial preferential flow.

A few of the known multi-well microfiltration arrangements provide acollection plate, for placement beneath a sample-well plate, having aplurality of closed-bottom collection wells corresponding to the samplewells. Generally, the collection of filtrate takes place uponapplication of a vacuum to pull the mobile phase through each well. Withmost of these arrangements, attempts to separately collect the filtratefrom each sample well have suffered from unreliable results due tocross-contamination between the wells of the collection plate. Aprincipal cause of such cross-contamination relates to the production ofaerosols as the filtrate leaves the drip directors. The aerosols canreadily disperse and travel to neighboring collection wells. Inaddition, aerosols may expose technicians to potentially pathogenicmicroorganisms, and the like, which may be present in the samples.

Cross-contamination due to aerosol formation is exacerbated by thetypical flow pattern induced by the vacuum arrangements of such systems.Usually, the sample-well plate is mounted above the collection plate,and the collection plate, in turn, sits in a vacuum chamber. Uponevacuation of the chamber, solution within each well is drawn downthrough the filter element toward a respective collection well.Generally, the vacuum draws along flow pathways extending from withineach mini-column, through a respective drip director, and horizontallyacross the top of the collection plate until reaching one side of thecollection plate whereat the flow pathways turn downward toward an exitport. Except for those drip directors located directly adjacent the sideof the chamber having the exit port, substances (e.g., entrainedaerosols, gases, etc.) pulled along each vacuum flow pathway from eachdrip director must pass by neighboring collection wells as they travelacross the top of the collection plate. Unfortunately, aerosols fromfiltrate exiting one drip director can become entrained in the flowacross the collection plate and make its way over into neighboringwells.

The potential for cross-contamination is particularly high when theupper sample-well and drip-director plates are removed from thecollection plate. Pendent drops of filtrate remaining on the dripdirectors can inadvertently fall into neighboring wells as the dripdirectors are moved over the collection plate. With standard multi-wellplates, a concerted, manual “touch-off” of all such pendent drops to theinner sides of respective collection wells is difficult, if notimpossible, due to the great number of wells. Application of a strongvacuum below the drip directors, in an attempt to pull such pendentdrops down and away from the drip directors, can atomize the pendentdrops, resulting in the related problem of contamination by aerosolformation, discussed above.

There is, thus, a need for a multi-well microfiltration arrangement thatprovides for the separate collection of filtrate from each well, whileavoiding cross-contamination due to aerosol formation and/or pendentdrops.

SUMMARY OF THE INVENTION

One aspect of the present invention provides a microfiltration apparatusfor processing a plurality of fluid samples.

According to one embodiment, the microfiltration apparatus of theinvention includes a first plate having a plurality of columns and asecond plate having a plurality of discharge conduits. Each of thecolumns has a first inner bore defining a lumen within the column and anend region for receiving a filter medium within the column. The columnend region defines a second inner bore having a diameter greater thanthat of the first inner bore and a transition region that joins thesecond inner bore to the first inner bore. A filter medium for filteringsample is positioned within each column end region, adjacent thetransition region. Each discharge conduit has an upstanding upper endregion aligned with and received within a corresponding column endregion so as to form a substantially fluid-tight interface therebetween.The discharge conduit upper end region has a terminal rim region forsupporting a circumferential region of the filter medium such that eachfilter medium is held between a column transition region and theterminal rim region of a corresponding discharge conduit.

In one embodiment, the transition region of each column has an annulartapered portion. The circumference of the annular tapered portiondecreases in a substantially constant fashion along a direction from thesecond inner bore to the first inner bore. In a related embodiment, aline running along the tapered portion, longitudinally with respect tothe column, forms an acute angle with a plane perpendicular to alongitudinal axis of the column and intersecting the column through ajunction of the transition region with the second inner bore.

The acute angle, in one embodiment, is within the range of about 30-70degrees. Preferably, the acute angle is within the range of about 30-60degrees. In one particular embodiment, the acute angle is about 45degrees.

According to one embodiment, the terminal rim region of each dischargeconduit contacts no more than about 15%, and preferably less than about10%, and more preferably less than about 5% of the bottom surface areaof a respective filter medium.

One embodiment provides a plurality of fin-like support buttresses ineach of the discharge conduits. In this embodiment, each of the supportbuttresses has an elongated, narrow, uppermost surface that issubstantially coplanar with a plane defined by the terminal rim regionof a respective discharge conduit. In a related embodiment, thehorizontal cross-sectional area of an upper region of each supportbuttress decreases in a direction extending towards its uppermostsurface in a fashion such that the intersection of the uppermost surfacewith the plane of the terminal rim region is substantially tangential innature, forming a line.

According to another embodiment, the microfiltration apparatus isprovided with a gas-permeable matrix comprised at least in part of aporous hydrophilic polymer material. The matrix is attached to thesecond plate on a face opposite the first plate. Also in thisembodiment, the matrix circumscribes a plurality of the dischargeconduits.

A further embodiment provides means for shifting the first and secondplates in either of two directions from a reference “home” positionalong a generally horizontally extending axis, and then returning theplates back to the reference “home” position. The shifting means caninclude a stepper motor disposed in mechanical communication with theplates such that angular rotation of the stepper motor induces linearmotion of the plates.

In accordance with another embodiment, vacuum means are provided fordrawing adherent drops of fluid hanging from the discharge conduits in adirection away from the collection wells and up into the dischargeconduits.

In another of its aspects, the present invention provides a method forforming a plurality of microfiltration wells. In one embodiment, a sheetof filter medium is positioned between a first plate containing aplurality of columns and a second plate having a plurality of dischargeconduits. Each of the columns has a first inner bore defining a lumenwithin the column and an end region defining a second inner bore havinga diameter greater than that of the first inner bore and a transitionregion that joins the second inner bore to the first inner bore. Each ofthe discharge conduits has an upstanding upper end region facing thefirst plate and aligned with a corresponding column end region. Theplates are pressed together in a manner effective to punch portions ofthe filter medium from the sheet to afford a filter medium plug situatedwithin the end region of each column in abutment with the columntransition region and a terminal rim region of a corresponding dischargeconduit upper end region.

The method of the invention also provides for the compression-fitsealing is of each filter element. In one embodiment, compression ofeach filter element between the column transition region and a terminalrim region of a corresponding discharge conduit upper end region servesto secure and seal the filter element to an inner sidewall of thecolumn.

In another embodiment, the method further includes the step of securingthe first plate to the second plate. The securing step can be effectedby forming a bond, such as an ultrasonic weld, between an inner sidewallof each second inner bore and an outer circumferential surface of arespective upper end region.

A further aspect of the present invention provides a microfiltrationapparatus for processing a plurality of fluid samples.

In one embodiment, the apparatus includes a first plate having aplurality of columns. Each of the columns contains, at one end thereof,a filter element and a fluid discharge conduit beneath the filterelement. A second plate is spaced apart from the first plate by acavity. The second plate has a plurality of receiving or collectionwells that are aligned with the columns for receiving sample fluid fromthe discharge conduits. The second plate is also provided with aplurality of vents adjacent the collection wells. A gas-permeable matrixis positioned in the cavity between the first plate and the second plateso as to fill the space between the confronting surfaces of the twoplates. The matrix laterally surrounds the region between at least onedischarge conduit and an aligned collection well. The matrix iseffective (i) to permit a vacuum drawn from beneath the second plate toextend, via the vents, to a region above the second plate and to thecolumns, thereby drawing fluid from the columns into the collectionwells and (ii) to obstruct movement of aerosols across the top of thesecond plate, thereby limiting cross-contamination between wells.

According to one embodiment, the matrix is a continuous sheet having aplurality of openings permitting the passage of filtrate from eachdischarge conduit to a respective collection well. Each one of thedischarge conduits can extend at least partially into a respective oneof the openings. Further, the matrix can extend over a plurality of thevents. In one embodiment, the matrix is comprised of a poroushydrophilic polymer material, such as ethyl vinyl acetate (EVA) or thelike.

In one embodiment, the collection wells are arranged in a rectangulararray having at least eight wells (e.g., 8, 12, 24, 48, or 384 wells).In one preferred arrangement, the second plate is provided with at leastone vent for every four collection wells, and the vents are arrangedsuch that a vent is located between each collection well and at leastone adjacent collection well. For example, a vent may be providedbetween each collection well and at least one diagonally adjacentcollection well of the array.

According to one embodiment, each of the columns has a first inner boredefining a lumen within the column and an end region defining a secondinner bore, having a diameter greater than that of the first inner bore,and a transition region that joins the second inner bore to the firstinner bore. Each of the discharge conduits has an upstanding upper endregion aligned with and received by a corresponding column end region soas to form a substantially fluid-tight interface therebetween. Thedischarge conduit upper end region has a terminal rim region forsupporting a circumferential region of the filter element such that eachfilter element is held between a column transition region and theterminal rim region of a corresponding discharge conduit.

In another embodiment, means are provided for shifting the first platein either of two directions from a reference “home” position along agenerally horizontally extending axis, and then returning the plate backto the reference “home” position. The shifting means can include astepper motor disposed in mechanical communication with the plate suchthat angular rotation of the stepper motor induces linear movement ofthe plate.

In a further embodiment, vacuum means are provided for drawing adherentdrops of fluid hanging from the discharge conduits in a direction awayfrom the collection wells and up into the discharge conduits.

Another aspect of the present invention provides a method for separatelycollecting filtrate from an array of microfiltration wells in acorresponding array of closed-bottom collection wells held by acollection tray situated below the microfiltration-well array.

In one embodiment, the method includes the steps of:

(A) placing a fluid sample in a plurality of the microfiltration wells;

(B) drawing a vacuum along pathways extending from each microfiltrationwell downward through a plane defined by an upper surface of thecollection tray at a point at or adjacent a corresponding collectionwell to a region beneath the collection tray, thereby causing a filtrateto flow from each microfiltration well and to collect in correspondingcollection wells; and

(C) obstructing aerosols formed from the filtrate at any onemicrofiltration well from moving across the upper surface of thecollection tray to a non-corresponding collection well, thereby limitingcross-contamination.

According to one embodiment, each vacuum pathway passes through agas-permeable matrix disposed in a cavity between themicrofiltration-well array and the collection-well array. Thegas-permeable matrix can be comprised of a porous hydrophilic polymermaterial, such as ethyl vinyl acetate (EVA) or the like. In onepreferred arrangement, the gas-permeable matrix circumscribes the regionbetween each microfiltration well and a corresponding collection well.

In one embodiment, the vacuum pathways pass through the plane of thecollection-tray upper surface by way of vents that traverse thecollection tray proximate each of said collection wells. Also in thisembodiment, the gas-permeable matrix covers the vents.

In another embodiment, each of the vacuum pathways extends from onemicrofiltration well into a respective collection well prior to passingthrough the vents.

In a further embodiment, wherein a collection tray having open-bottomwells is used, the vacuum pathways pass through the plane of thecollection-tray upper surface and then down and out of the open bottomsof the wells.

The microfiltration wells comprise, according to one embodiment, a firstplate having a plurality of columns and a second plate having aplurality of discharge conduits. Each column has a first inner boredefining a lumen within the column and an end region for receiving afilter medium within the column. The end region defines a second innerbore having a diameter greater than that of the first inner bore and atransition region that joins the second inner bore to the first innerbore. A filter medium for filtering sample is positioned within eachcolumn end region, adjacent the transition region. Each dischargeconduit has an upstanding upper end region aligned with and receivedwithin a corresponding column end region so as to form a substantiallyfluid-tight interface therebetween. The discharge conduit upper endregion has a terminal rim region for supporting a circumferential regionof the filter medium such that each filter medium is held between acolumn transition region and the terminal rim region of a correspondingdischarge conduit.

In one embodiment, the method includes the additional steps of:

(i) touching-off, in a substantially simultaneous fashion, adherentdrops of fluid hanging from the bottom of each microfiltration well toan inner sidewall of a respective collection well; and

(ii) drawing adherent drops of fluid hanging from the discharge conduitsin a direction away from the corresponding collection wells and up intothe discharge conduits.

In another of its aspects, the present invention provides an apparatusfor avoiding cross-contamination due to pendent drops of fluid hangingfrom a plurality of discharge conduits disposed in an array above acorresponding array of collection wells.

According to one embodiment, the apparatus includes:

(i) a carriage configured to carry one of the arrays and adapted forlinear reciprocal motion in either of two directions along a first,generally horizontal, axis from a neutral position whereat the arraysare substantially axially aligned;

(ii) a stepper motor:

(iii) a linkage assembly mechanically communicating the stepper motorwith the carriage such that each rotational step of the stepper motorinduces movement of the carriage a given distance from the neutralposition in one of the two directions depending upon the direction ofangular rotation of the motor, thereby effecting relative motion betweenthe discharge-conduit array and the collection-well array such thatpendent drops of fluid hanging from the discharge conduits aresimultaneously touched-off to inner sidewalls of correspondingcollection wells; and

(iv) a compression spring mounted within the linkage assembly in amanner permitting the spring (a) to provide a predetermined amount ofresistance to movement of the carriage from the neutral position, and(b) to compensate or absorb some of the linear overshoot due to excessangular rotation of the motor beyond the amount required to move thedischarge conduits into firm abutment with the inner sidewalls of thecollection wells.

In one embodiment, a vacuum chamber communicates with thedischarge-conduit array from a side thereof opposite the collection-wellarray. Evacuation of the vacuum chamber is effective to urge pendentdrops of fluid hanging from the discharge conduits in a direction awayfrom the collection wells and into the discharge conduits.

In one preferred embodiment, the carriage is configured to carry thedischarge-conduit array, while the collection-well array remainsstationary. A vertical positioning assembly can be disposed on thecarriage to support the discharge-conduit array for linear movementalong a second, generally vertical, axis between a lowered positionwhereat the discharge conduits extend down into respective collectionwells and an elevated position whereat the discharge conduits clear thecollection wells.

Still a further aspect of the present invention provides a method foravoiding cross-contamination due to pendent drops of fluid hanging froma plurality of discharge conduits disposed in an array above acorresponding array of closed-bottom collection wells.

In one embodiment, the method includes the steps of:

(i) touching-off, in a substantially simultaneous fashion, pendent dropsof fluid hanging from the discharge conduits to inner sidewalls ofrespective collection wells; and

(ii) drawing pendent drops of fluid hanging from the discharge conduitsin a direction away from the corresponding collection-well array andinto the discharge conduits.

The touching-off step can be carried out by shifting thedischarge-conduit array along a plane substantially orthogonal to thelongitudinal axes of the collection wells, while the collection wellsare maintained in a substantially fixed position. In one embodiment,each of the discharge conduits is shifted into contact with one sidewallportion of a respective collection well, and then is shifted intocontact with another, laterally opposing sidewall portion of therespective collection well.

One embodiment provides a stepper motor in mechanical communication withthe discharge-conduit array such that angular rotation of the steppermotor induces linear motion of the discharge conduits. In thisembodiment, stepping of the stepper motor causes the discharge-conduitarray to shift.

The step of drawing pendent drops of fluid can be effected byestablishing a reduced pressure (a vacuum) above the discharge conduits.

In one embodiment, an upstanding upper end region of each of thedischarge conduits is received within a respective column, therebyforming an array of microfiltration wells. Each column has a first innerbore defining a lumen within the column and an end region defining asecond inner bore having a diameter greater than that of the first innerbore and a transition region that joins the second inner bore to thefirst inner bore. A filter element is disposed in each column, betweenthe transition region of the column and the upper end region of arespective discharge conduit.

In another of its aspects, the present invention provides a removablecover for isolating a plurality of samples separately contained in anarray of closed-bottom wells supported in a collection tray.

According to one embodiment, the cover includes a substantially rigid,rectangular shell portion having a top surface, a bottom surface and acircumferential side-edge region. A plurality of reversibly expandable,tubular sleeves are provided on the top surface of the shell portion. Aresiliently compliant undersurface is secured to the bottom surface ofthe shell portion. A plurality of resiliently deflectable, elongatedside arms project below the bottom surface from opposing side-edgeregions of the shell portion. In its normal (unstressed) state, eachside arm is positioned substantially perpendicular to a plane defined bythe bottom surface. An inwardly directed catch is formed at an end ofeach side arm, distal from the shell portion. The arms, and associatedcatches, are useful for releasably snap-locking the cover over the wellsof a collection tray.

In one embodiment, the undersurface of the cover includes a plurality ofdownwardly convex nodules (half-dome features) disposed in an arraycomplementary to the collection-well array. Each nodule is adapted tofit over a corresponding well when the cover is secured over thecollection tray.

A further aspect of the invention provides a method for covering anarray of open-top wells held in a collection tray.

According to one embodiment, the method is carried out in asubstantially automated fashion using (i) a support structure adaptedfor movement along a generally horizontal plane (x/y direction) and (ii)a plurality of elongated, parallel rods depending from the support andadapted for movement along their respective longitudinal axes (ydirection). Initially, the rods, while disposed in a retracted positionadjacent the support, are positioned over a cover member. Two of therods are then extended away from the support (y direction) so that theirend regions become wedged in respective cavities formed along the top ofthe cover, while two rods are maintained in the retracted position(i.e., with free end regions). The cover member is then lifted byretracting the wedged rods back toward the support. The support is thenmoved along the x/y direction so that the cover becomes positioned overthe collection tray. The wedged rods are then extended away from thesupport so that the cover is lowered onto the collection tray, over thewell openings. The free ends of two retracted rods are then extendeduntil they abut an upper region of the cover, thereby blocking upwardmovement of the cover, while the wedged rods are retracted away from thecover so that they are withdrawn (unwedged) from the cavities. As aresult, the cover is left resting on top of the collection tray over thewell openings.

From this position, the cover member can be releasably snap-locked to isthe collection tray. This can be effected, for example, by extending atleast one of the rods away from the support and into abutment with anupper region of the cover, thereby pressing the cover into lockingengagement with the collection tray. Another of the rods can be extendedaway from the support and into abutment with another upper region of thecover in order to prevent the cover from flipping up while being locked.

The method can be carried out, for example, with a cover having (i) anupper, substantially rigid shell portion, (ii) a lower, compliantundersurface secured to the shell portion, and (iii) means forreleasably locking the shell portion to the collection tray. Theundersurface of the cover can include, for example, a plurality ofdownwardly convex nodules (half-dome features), disposed in an arraycomplementary to the well array. Further, the shell portion can includea plurality of landing sites along its upper surface configured toreceive the lower end regions of the rods.

Still a further aspect of the invention provides a device for holding aplurality of rectangular, heat-sealable sheets.

In an exemplary embodiment, the device is comprised of a tray having asubstantially rectangular bottom surface, four upwardly divergentsidewalls extending from the bottom surface, and an uppercircumferential edge region defining a substantially rectangular opentop. A plurality of ribs run along each sidewall, spanning most of thedistance between the bottom surface and the upper circumferential edgeregion. Each of the ribs has a substantially linear surface that (i)faces an opposing sidewall and (ii) is substantially normal to a planedefined by the bottom surface of the tray.

According to one embodiment, a plurality of heat-sealable sheets,arranged in a vertical stack, is positioned in the tray such thatperipheral side-edge regions of the sheets are disposed in contact withthe substantially linear surface of each rib.

Another aspect of the present invention provides a method of sealing arectangular, heat-sealable sheet over an array of wells held in acollection tray.

In one embodiment, the method includes the steps of (i) picking up aclear heat-sealable sheet; (ii) placing the sheet over open upper endsof the wells; and (iii) pressing a conformable heated surface againstthe sheet, from a side opposite the collection tray, with sufficientpressure such that the sheet is heat-sealed to the collection tray overthe open upper ends of the wells. Further according to this embodiment,the conformable heated surface is pressed against the sheet using aplurality of spaced-apart elongated rods, disposed substantially normalto an upper surface of the collection plate. The rods can depend from asupport structure positioned above the collection plate.

These and other features and advantages of the present invention willbecome clear from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

The structure and manner of operation of the invention, together withthe further objects and advantages thereof, may best be understood byreference to the following description taken in conjunction with theaccompanying drawings, in which identical reference numerals identifysimilar elements, and in which:

FIG. 1 is a perspective view of a multi-well microfiltration apparatusconstructed in accordance with an embodiment of the present invention.

FIG. 2 is an exploded view of the multi-well microfiltration apparatusof FIG. 1.

FIG. 3 is a partial side-sectional view of the multi-wellmicrofiltration apparatus of FIGS. 1 and 2.

FIG. 4 shows, in enlarged detail, one microfiltration well from thesectional view of FIG. 3.

FIG. 5 is a partial side-sectional view showing a microfiltration wellconstructed in accordance with an embodiment of the present invention.

FIG. 6 is an exploded view of a microfiltration well showingmembrane-support structure in the form of three fin-like supportbuttresses constructed in accordance with an embodiment of the presentinvention.

FIG. 7 is an elevational view from one end of a carriage assembly foreffecting relative motion between the drip directors of a drip-directorplate and the collection wells of a collection plate, according to anembodiment of the present invention.

FIG. 8 is a partially exploded, perspective view showing a carriageassembly for effecting relative motion between the drip directors of adrip-director plate and the collection wells of a collection plate,according to an embodiment of the present invention.

FIGS. 9(A)-9(C) are side cross-sectional views showing a touch-offoperation whereby a plurality of drip directors is laterally shifted tothe right and to the left such that the drip director outlet regionssimultaneously abut inner sidewalls of a plurality of correspondingcollection wells.

FIG. 10(A) is a partially schematic top plan view showing aspring-loaded touch-off mechanism in its normal, or neutral, position.

FIG. 10(B) is a partially schematic top plan view showing thespring-loaded touch-off mechanism of FIG. 10(A) in a first, shiftedposition.

FIG. 10(C) is a partially schematic top plan view showing thespring-loaded touch-off mechanism of FIGS. 10(A)-10(B) in a second,shifted position.

FIG. 11 is a perspective view of a cover member, having an array ofresiliently flexible half-dome features on its lower face, disposed overa multi-well tray, in accordance with an embodiment of the presentinvention.

FIG. 12 is another perspective view of the cover member of FIG. 12,showing a plurality of sites on the cover's upper surface for receivingthe lower end regions of elongated, fluid-handling fingers of afluid-handling robot positioned above the tray, in accordance with anembodiment of the present invention.

FIG. 13 is a perspective view showing the cover of FIGS. 11 and 12disposed over the openings of a multi-well tray and releasablysnap-locked to the multi-well tray, according to an embodiment of theinvention.

FIGS. 14(A) and 14(B) are enlarged, perspective and side-sectionalviews, respectively, showing a releasable snap-locking assembly forsecuring a cover of the invention to a multi-well tray, according to anembodiment of the present invention.

FIG. 15 is a perspective view showing an assembly for releasablysecuring a cover of the invention to a multi-well tray, according to afurther embodiment of the invention.

FIG. 16 is a perspective view showing an automated high-throughputsample preparation workstation, including, for example, amicrofiltration apparatus, cross-contamination control arrangements,collection-well covering and heat-sealing assemblies, and associatedcomponents and reagents, in accordance with the teachings of the presentinvention.

FIG. 17 is a perspective view of an automated station for applyingheat-sealable sheets over the wells of a multi-well tray, in accordancewith an embodiment of the present invention.

FIG. 18 is a perspective view showing a tray or bin for holding a stackof heat-sealable sheets, constructed in accordance with an embodiment ofthe present invention.

FIGS. 19(A) and 19(B) are enlarged, perspective and side-sectionalviews, respectively, showing a releasable snap-locking assembly forsecuring a tray or bin, such as shown in FIG. 18, to a frame assemblysituated, for example, at a heat-sealing station such as shown in FIG.17, in accordance with one embodiment of the present invention.

FIGS. 20 to 23 are perspective views illustrating various features, aswell as the operation of, the automated heat-sealing station of FIG. 17,according to an embodiment of the present invention.

FIG. 24 is a perspective view, with portions broken away, of a heatableplaten assembly as used, for example, in the heat-sealing station ofFIGS. 17 and 20-23, according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The following discussion of the preferred embodiments of the presentinvention is merely exemplary in nature. Accordingly, this discussion isin no way intended to limit the scope of the invention, application ofthe invention, or the uses of the invention.

FIGS. 1-3 show, in perspective, exploded and partial side-sectionalviews, respectively, an embodiment of a multi-well microfiltrationapparatus constructed in accordance with the present invention. In theassembly stage of manufacture, a filter sheet or membrane, indicated inFIG. 2 by the reference numeral 8, is located between a column tray, orplate, 10 having an array of open-bottom mini-columns, such as 12, and adrip-director tray, or plate, 14 having an array of drip directors, suchas 16, corresponding to the mini-columns. Upon registering and matingmini-columns 12 with drip directors 16, an array of microfiltrationwells are formed, denoted generally in FIG. 3 by the reference numeral18, each having a discrete filter element or medium (e.g., a plug, disc,or the like), such as 8 a and 8 b, positioned therein. The inner wallsof each mated mini-column/drip-director pair bound a flow pathway whichextends downward through the well 18.

As shown in FIGS. 2 and 3, each microfiltration well has an interiorregion, or lumen, that is substantially circular in horizontalcross-section. It should be appreciated, however, that microfiltrationwells of any desired geometrical cross-section (e.g., oval, square,rectangular, triangular, etc.) could be used. Similarly, the wells maybe of any desired shape when viewed along their longitudinal axes, e.g.,straight, tapered or other shape. In one embodiment, the walls of eachwell have a slight outward taper (i.e., the well diameter increases)along the direction extending from the well's upper, loading end towardthe filter medium.

The plates of the microfiltration apparatus may be constructed of anysubstantially rigid, water-insoluble, fluid-impervious material that issubstantially chemically non-reactive with the assay samples. The term“substantially rigid” as used herein is intended to mean that thematerial will resist deformation or warping under a light mechanical orthermal load, although the material may be somewhat elastic. Suitablematerials include acrylics, polycarbonates, polypropylenes andpolysulfones. Also, it should be noted that the terms “tray” and “plate”are used synonymously and interchangeably herein.

Optionally, the fluid-contacting surfaces of the drip directors can becomprised of a material and/or provided with a coating that renders suchsurfaces hydrophobic, reducing the potential for cross-contamination.For example, low surface-energy materials could be used in formingand/or coating the drip directors. Of course, such materials should becompatible with the assay samples.

The plates may be formed by any conventional means, injection moldingbeing a particularly convenient technique. One embodiment of theinvention contemplates the use of injection molded rectangular plasticplates, the length and width of which conform to the commonly usedstandard of 5.03″×3.37″ (127.8 mm and 85.5 mm). In the embodiment ofFIGS. 1-3, the wells are formed integrally with such a plate, arrangedin a 12×8 regular rectangular array spaced 0.9 cm center-to-center.Alternatively, the wells can be formed as discrete units (not shown)interconnected by plastic webbing to provide an array. In anotherembodiment, the wells are provided in the form of strips (not shown).For example, a plurality of wells could be disposed in a row withadjacent wells connected to one another by any suitable means, e.g.,frangible plastic webs. A plurality of strips could then be arrangedside-by-side within a frame designed to hold such strips. For example,twelve 8-well strips could be placed side-by-side in a rectangular frameto form a 96-well array. In a further embodiment, each well is formed asa discrete unit removably positioned within a respective opening formedin a support plate (not shown). For example, a tray could be providedwith a 12×8 array of circular openings in which cylindrical wells arereceived and held, in a fashion similar to test-tubes held in aconventional test-tube rack.

Although the illustrated embodiments show arrangements configured inaccordance with the popular 96-well format, the invention alsocontemplates any other reasonable number of wells (e.g., 12, 24, 48,384, etc.) disposed in any suitable configuration.

With reference once again to FIGS. 1-3, an upper vacuum chamber 20 issituated above column plate 10. Upper vacuum chamber 20 is adapted formovement between (i) a mounted position, whereat four dependingcircumferential walls, denoted as 20 a, form a substantially airtightseal with an upper, peripheral surface of column plate 10 via aninterposed resilient gasket 21, and (ii) a retracted position, whereatchamber 20 is spaced apart from column plate 10. The hollow interior ofchamber 20 is pneumatically connectable to an external vacuum source viaa hosecock 23 extending through the top of chamber 23. A reducedpressure can be established above the sample wells by bringing chamber20 to its mounted position atop column plate 10 and then evacuatingchamber 20.

In some situations, it may be desirable to establish an increasedpressure above the sample wells (e.g., to facilitate the flow of samplesthrough the filter media and out of the wells via the lower dischargeconduits). In such cases, chamber 20 can be pressurized by way of asuitable pressure source (e.g., a pump).

A receiving, or collection, plate 24 is located below drip directorplate 14. Collection plate 24 includes an upper planar surface, denotedas 25, and an array of closed-bottom wells, such as 26, dependingtherefrom. The collection-well array corresponds to the drip-directorarray, permitting the separate collection of filtrate from each samplewell. The collection plate is adapted to fit inside an open reservoir ofa lower vacuum chamber, denoted as 29, with the collection wellsextending down into the reservoir.

Apertures or vents, such as 28, extend through the upper planar surface25 of collection plate 24. For reasons that will become apparent, atleast one aperture should be located adjacent each collection well. Theapertures 28 permit fluid communication between the regions above andbelow the plate 24. By this construction, a vacuum drawn from beneaththe collection plate will extend to the regions above the plate andinside the wells.

Although not shown in the figures, the present invention also provides aplate like collection plate 24, except having open-bottom wells asopposed to the closed-bottom wells of plate 24. Otherwise, the plate ofopen-bottom wells is configured like collection plate 24. That is, theplate of open-bottom wells is provides structure for effectivelycarrying out filtrations and/or washings, while avoidingcross-contamination. However, instead of separately collecting filtratein the various wells, the filtrate passes through the wells and out ofthe open bottoms. It is contemplated that the plate of open-bottom wellswill be used in a manner like that described herein for plate 24, exceptthat the situation will not call for the separate collection offiltrate. For example, the plate of open-bottom wells is particularlyuseful in performing intermediate washings. As used herein, “collectionplate” and “receiving plate” are used synonymously and interchangably,with either term referring to a plate, intended for placement beneath adrip-director array, having either open-bottom wells or closed-bottomwells, as appropriate for the task at hand. Where the separatecollection of filtrate is to take place, it is understood that the wellsare of a closed-bottom type. Optionally, a collection plate havingopen-bottom wells may be formed without vent features (such as 28), asthe vacuum can flow directly down and out through the bottom of eachwell.

A cross-flow restrictor (also referred to as an aerosol guard), denotedas 30, which is generally pervious to gases but substantially imperviousto aerosols, is interposed between the upper surface of collection plate24 and the lower surface of drip-director plate 14. In the illustratedembodiment, cross-flow restrictor 30 has a plurality of passages, suchas 32, arranged in an array complementing the collection-well anddrip-director arrays. Passages 32 permit filtrate to pass from each dripdirector 16 to a corresponding collection well 26. In the illustratedarrangement, each drip director 16 extends through a respective passage.Except for such passages, cross-flow restrictor 30 substantially fillsthe area between the confronting faces of the drip-director andcollection-well plates (14, 24).

Preferably, means are provided for supporting the assembled mini-columnand drip-director plate arrangement, and assisting in the formation ofan airtight seal between this arrangement and the lower vacuum chamber29. In the illustrated embodiment, a rectangular carriage frame, denotedas 38, is configured to support the mini-column and drip-director plateassembly. Clamps 34, 36 are pivotally mounted about generally verticallyextending axes at opposing ends of frame 38. Clamps 34, 36 are operableto engage and hold the column and drip-director assembly on frame 38,with a lower peripheral edge 40 of the column and drip-director plateassembly pressed against a gasket 42 disposed on the upper surface offrame 38 about the frame's central opening.

A spring-loaded centering pin, such as 37 and 39, may extend througheach clamp 34, 36. In the embodiment of FIG. 3, centering pin 37 has ashank that is urged by a spring 41 to sit within a complementary recessor depression 43 formed in a sidewall of column plate 10. In anotherembodiment (not shown), three spring-loaded centering pins are employed,with two pins located at positions on a long side of the arrangement andone pin located at a position on a short side, together operable to pushthe tray against a corner. In this way, the components can be readilycentered (on axis).

A stepped gasket, indicated generally at 44, is disposed adjacent alower surface of frame 38 about the frame's central opening. Gasket 44has (i) an upper, inwardly projecting flap portion, denoted as 44 a,having a lower surface adapted to engage an upwardly projecting ridge 48disposed about the periphery of collection plate 24, and (ii) a lowerflap portion, denoted as 44 b, that extends diagonally downward andoutward for engaging an upper surface 50 surrounding the open reservoirof lower vacuum chamber 29. A central plateau region of stepped gasket44, denoted as 44 c, is secured to frame 38 by any suitable means. Forexample, central plateau region 44 c can be attached using an adhesiveand/or fasteners. In one embodiment, gasket 44 is interposed betweenframe 38 and a rectangular clamping frame (not shown). In thisembodiment, the rectangular clamping frame is disposed adjacent theplateau region 44 c of gasket 44, on a side of gasket 44 opposite frame38. The clamping frame is then snugly secured to frame 38 using threadedfasteners that pass through aligned passages (not shown) formed in theclamping frame and gasket, and are received in internally threaded boresextending partially into frame 38 from the frame's lower surface.Together, upper gasket 42 and lower gasket 44 assist in formingsubstantially airtight seals between (i) the upper microfiltration wellassembly and the carriage frame, and (ii) the carriage frame and thelower vacuum chamber assembly, respectively.

The gaskets (21, 42, and 44) may be formed of any deformable, resilient,substantially inert material capable of forming a seal. Examples of suchmaterials are silicone, rubber, polyurethane elastomer and polyvinylchloride. The thickness of each gasket is not critical, provided onlythat it is sufficient to form a seal. Typical gasket thicknesses willrange from about 1 mm to about 5 mm.

Once appropriate airtight seals are formed, evacuation of lower vacuumchamber 29 establishes a substantially uniform pressure drop over all ofthe sample wells 18, permitting a plurality of individual samples (e.g.,up to ninety-six in the illustrated embodiment) to be processedsimultaneously on the membrane of choice.

Those skilled in the art will recognize that the choice of filter mediumwill depend on the intended use of the well. For example, the filtermedium might serve as a size exclusion filter, or it could serve as asolid phase interacting with a species in the liquid phase to immobilizesuch species upon contact, such as an immunological interaction or anyother type of affinity interaction. Examples of suitable filtersinclude, but are not limited to, those of nitrocellulose, regeneratedcellulose, nylon, polysulfone, glass fiber, blown microfibers, andpaper. Suitable filters are available from a variety of sources, e.g.,Schleicher & Schuell, Inc. (Keene, N. H.) and Millipore Corp. (Bedford,Mass.).

Additional examples of suitable filters include microfiber filters ofultra-pure quartz (SiO₂), e.g., as manufactured by Whatman, Inc.(Tewksbury, Mass.) and sold under the trademarks QM-A and QM-B. QM-Afilters are about 0.45 mm thick and retain particles of about 0.6 μm.QM-B filters are of the same composition as QM-A, but are two timesthicker and therefore provide a longer tortuous path to flow. In oneembodiment, a quartz or glass filter element is fired (e.g., at about400° C.) prior to placement in a microfiltration well in order to reduceparticle generation, thereby reducing the potential for clogging of thedrip directors.

In another embodiment the filter medium is a porous element that acts asa frit, serving to contain a column packing material (e.g.,reversed-phase or size-exclusion packings).

Certain aspects of the invention that address the aforementionedproblems pertaining to (i) cross-contamination due to wicking across acommon filter sheet and (ii) individual filter elements entrappingsample constituents within substantial dead volumes will now bedescribed in greater detail.

One microfiltration well from the sectional view of FIG. 3 is shown inenlarged detail in FIG. 4. Mini-column 12 and drip director 16 areaxially aligned and mated, with an upwardly protruding portion of dripdirector 16 snugly received within the lower region of the mini-columnlumen to form a substantially fluid-tight well 18.

Means are provided for holding the drip director and mini-columntogether. In one embodiment, ultrasonic welds or bonds (not shown)formed along an annular region of contact, as designated in FIG. 4 bythe reference numeral 48, hold mini-column 12 and drip director 16together. It should be appreciated that such a weld or bond helps toensure a fluid-tight interface between these elements. In anotherembodiment, the mini-column 12 and drip director 16 are held together bya tongue-in-groove arrangement (not shown) formed along confrontingsurfaces of plates 10 and 14. For example, the column plate could beformed with deep scoring or grooves along its lower surface,circumscribing each well. The upper surface of the drip-director platecould be provided with upwardly projecting ridges, disposed in a patterncomplementary to the groove pattern of the column plate and configuredto snap-fit within the grooves. Alternatively, the mating of the dripdirectors with the mini-columns may be sufficiently snug as to hold theplates together solely by frictional engagement.

Means are provided for holding each individual filter element within arespective assembled microfiltration well. In this regard, each filterelement is disposed within the mini-column lumen so that a portion ofits peripheral edge is held between (i) a constricted-diameter regionwithin the lower portion of the mini-column and (ii) an upper portion ofthe drip director. The central region of the filter element extendsfully across the mini-column lumen.

In the embodiment of FIG. 4, mini-column 12 is formed with a bore 12 aand a counterbore 12 b, the latter extending upwardly from themini-column's lower end or lip 12 c. Between the bore 12 a andcounterbore 12 b, lies a transition region. The transition regionprovides a constricted-diameter region, or shoulder, within themini-column lumen capable of cooperating with an upper portion of acorresponding drip director to maintain the filter element in place. Thejunctions of the transition region with the bore and counterbore may beof any suitable shape. For example, such junctions could take the shapeof a hard angle or comer, or alternatively, they could take the shape ofa smooth curve. Further, the transition region itself, between suchjunctions, may be of any shape, e.g., flat, curved, stepped, or anycombination thereof, provided only that a suitable constricted-diameterregion is provided in the mini-column lumen for contacting an upper edgeregion of the filter element.

In one preferred embodiment, depicted in FIG. 4, the transition regionbetween bore 12 a and counterbore 12 b defines an internal, annularshoulder, denoted as 12 d. In this embodiment, each of the junctions ofshoulder 12 d with bore 12 a and counterbore 12 b defines a hard angleor corner. Between such junctions, the shoulder 12 d takes the form ofan annular wall having a substantially constant taper, with a decreasingcircumference along the direction from counterbore 12 b to bore 12 a.Longitudinally, the surface of shoulder 12 d is oblique to the surfacesof bore 12 a and counterbore 12 b. Preferably, the surface of shoulder12 d forms an acute angle with a plane perpendicular to the minicolumn'scentral axis and extending through the junction of shoulder 12 d withcounterbore 12 b. In one embodiment, this angle, denoted as a in FIG. 4,falls within the range of about 30-85 degrees; and is preferably withinthe range of about 60-85 degrees.

Drip-director 16 is configured to facilitate elution of a mobile phasefrom the well by funneling it toward a lower opening. In the embodimentof FIG. 4, drip director 16 includes (i) an annular edge or rim 16 adisposed above the plane of the upper surface of drip-director plate 14,(ii) depending convergent sidewalls 16 b, and (iii) a downspout oroutlet port 16 c disposed below the plane of the lower surface ofdrip-director plate 14. The downwardly sloping, inner surface of theconvergent sidewalls 16 b, between rim 16 a and outlet port 16 c,defines a conical and/or horn-shaped cavity at the lower region of thewell lumen.

As previously mentioned, an upper portion of drip director 16 providessupporting structure adapted to abut a lower peripheral edge region ofthe filter element. In the embodiment of FIG. 4, such structure takesthe form of upper, annular rim 16 a. The surface area of the uppermostregion of rim 16 a (i.e., the portion of rim 16 a that directlyconfronts, and is available to support, the lower peripheral edge regionof the filter element) may vary. In one preferred embodiment, theuppermost region of rim 16 a defines a narrow circular line. In thisembodiment, the contact between rim 16 a and filter element 8 a istangential in nature. That is, the region of contact between rim 16 aand filter element 8 a defines a very thin, circular line. Rim 16 acontacts no more than about 15%, and preferably less than about 10%, andmore preferably less than about 5% of the bottom surface area of thefilter element 8 a.

In the illustrated embodiment, the peripheral edge region of filterelement 8 a is preferably pinched or compressed between shoulder 12 dand rim 16 a in a manner effective to secure the filter element in placeand to press its circumferential side-edge against the inner surface ofthe column lumen. This arrangement discourages upward or downwardmovement of the filter element and prevents leakage around its edges.

FIG. 5 is a partial side-sectional view showing a microfiltration wellconstructed in accordance with one preferred embodiment of theinvention. Filter element 8 a is compressed between drip-director rim 16a and mini-column shoulder 12 d such that the membrane is securely heldin place. Further, the compression fit causes the outer circumferentialside-edge region of the filter element to press against the inner wallof the column lumen in a manner effective to avoid any bypassing offluid around the edges of the filter element. Shoulder 12 d extends intothe mini-column lumen at an angle a of about 45 degrees. Further, theuppermost surface area of rim 16 a is minimal, approaching that of acircular line, so that only the outermost perimeter of the filterelement's lower surface is in contact therewith.

With continued reference to FIG. 5, both the compression and the deadvolume have been estimated for a filter element in one suchmicrofiltration well using the computer-aided engineering package“Pro/ENGINEER” (Release 18), by Parametric Technology Corporation(Waltham, MA). The membrane compression for a 950 μm thick QM-B(Whatman, Inc., Tewksbury, Mass.) filter element having a diameter of6.88 mm is estimated to be only about 2.6 μl (area 52 in FIG. 5), andthe dead volume for such a filter element is estimated to be only about3 μl (area 54 in FIG. 5).

Beneath the filter element 8 a, the inner surface of the convergentsidewalls 16 b of drip director 16 define a cavity. The cavity isconfigured to expose the great majority of the filter element's lowersurface to open, or free, space. By providing such free space below thefilter element 8 a (i.e., volume between the drip director's convergentsidewalls 16 b and the lower surface of the filter element),preferential flow pathways are avoided.

In another embodiment, to prevent sagging or dislodgement of the filterelement into the cavity, the invention provides structure for supportingcentral points or regions of each filter element. For example, a supportbuttress may be disposed within the cavity of drip director 16 toprovide a resting point, edge or surface for one or more centrallylocated regions of the filter element's lower surface. Here, the term“central” refers to those portions of the filter element that s arelocated radially inward of the filter element's peripheral edges; andparticularly to those portions that are not held or pinched between aconstricted-diameter region in a mini-column and an uppermost rim of adrip director. In a preferred embodiment, the uppermost region of suchsupportive structure is substantially co-planar with the uppermostportion of the drip-director rim. It should be appreciated that suchstructure prevents downward sagging or dislodgment of the filter elementinto the cavity. This is particularly advantageous in connection withfilter elements lacking in substantial mechanical strength and/orrigidity.

In one preferred embodiment, shown in the exploded view of FIG. 6, suchsupportive structure takes the form of three fin-like supportbuttresses, denoted as 58 a-58 c, positioned radially and spacedequidistantly within the cavity of drip-director 16 about central outletport 16 c. It should be appreciated that any other reasonable number ofsupport buttresses, e.g., 4 or 6, may be employed instead. Smallportions of the lower surface of filter element 8 a rest on top ofelongated, narrow, uppermost surfaces or edges of the support buttresses58 a-58 c. Preferably, the support buttresses 58 a-58 c are configuredto support the filter element without introducing substantial deadvolume or preferential flow in the system. In this regard, the top ofeach support buttress, proximate the filter element, may be curved,arched, or angled so that the region of contact between the filterelement 8 a and each buttress is substantially along a line (i.e.,tangential in nature). Further, the profile of each support buttress isnarrow and streamlined along the direction of fluid flow.

In the illustrated embodiment, the support buttresses 58 a-58 c areformed integrally with the drip director 16. Alternatively, a pluralityof discrete support-buttress arrangements (not shown), formedindependently of the flow directors, may be removably positioned orpermanently affixed within respective drip directors.

Advantageously, the invention also provides a very efficient andcost-effective method for manufacturing the apparatus described herein.According to one embodiment, a sheet of filter material is positionedbetween a first plate, having a mini-column formed therein into which asample can be placed, and a second plate having a discharge conduit, ordrip director, with an outlet through which sample may egress. Theplates are positioned so that the mini-column is axially aligned withthe drip director. The plates are then pressed together so that anupwardly protruding portion of the drip director is snugly receivedwithin the lower region of the mini-column lumen. During the latteroperation, a flow pathway is formed, extending from within themini-column to the outlet of the drip director. Also during thecompression step, a piece of filter media is cut from the sheet andpositioned across a section of the flow pathway within the minicolumn.

The method of the invention is particularly advantageous forconstructing a multi-well microfiltration apparatus as detailed above.Therefore, the method of the invention will now be described withreference to the illustrated apparatus. Filter sheet 8 is interposedbetween the confronting surfaces of column plate 10 and drip-directorplate 14, as shown in FIG. 2. The plates 10, 14 are arranged so thateach mini-column 12 is in axial alignment with a corresponding dripdirector 16. The plates 10, 14 are then pressed together to achieve aconfiguration substantially as shown in FIG. 3. During the compressionstep, an upper annular rim 16 a of each drip director 16 acts as a dieto punch out a piece of filter media 8 a (e.g., in the form of a disc)from the filter sheet. Furthermore, compressing the drip director 16against the mini-column 12 secures the filter element in place withinthe mini-column lumen. As a result, an outer, peripheral edge portion offilter element 8 a is pinched between an upper, annular rim 16 a of dripdirector 16 and an internal, annular shoulder 12 d of mini-column 12.The drip director 16 and mini-column 12 are then secured together by anysuitable means. For example, an ultrasonic weld or a tongue-in-groovearrangement can hold the mini-columns 12 and drip directors 16 together,as discussed above.

A further aspect of the present invention pertains to a multi-wellmicrofiltration arrangement that provides for the flow of filtrate outof each well, while avoiding cross-contamination due to aerosols orsplattering.

As previously described, the collection-well array corresponds to thedrip-director array, with each drip director disposed directly over areceiving or collection well. The collection-well plate, in turn, isadapted to fit within an open reservoir of a lower vacuum chamber, withthe collection wells extending down into the reservoir. Uponestablishing a suitable vacuum in the lower chamber, filtrate will flowfrom each microfiltration well and into corresponding collection wells.In accordance with this aspect of the invention, means are provided fordiscouraging filtrate-associated aerosols and residues present at anyone well from traveling to, and potentially contaminating neighboringwells. Such means can include, for example, a cross-flow restrictor,also referred to as an aerosol guard, comprised of a substantiallyaerosol-impervious material, interposed in the region between the uppersurface of collection plate and the lower surface of drip-directorplate. While limiting the passage of aerosols and filtrate-associatedresidues, the cross-flow restrictor is adapted to permit a vacuum to bedrawn therethrough.

With particular reference to the embodiment of FIGS. 2 and 3, asheet-like cross-flow restrictor 30 is provided with an array,ofpassages 32 complementary to the collection-well and drip-directorarrays that permit filtrate to pass from each microfiltration well 18 toa corresponding collection well 26. Except for such passages, cross-flowrestrictor 30 substantially fills the area between the confronting facesof the drip-director and collection-well plates (14, 24). In this way,well-to-well movement of aerosols over the collection plate 24 issubstantially blocked. Consequently, the risk of cross-contaminationpresented by aerosol movement is substantially reduced. Additionally,aerosols formed at any one collection well that inadvertently passthrough the cross-flow restrictor (i.e., those that are not effectivelyblocked or trapped) will be pulled by the vacuum source through anadjacent aperture 28 down to the region below plate 24 without passingover the openings of neighboring collection wells, as described morefully below.

Embodiments of the present invention contemplate attachment of thecross-flow restrictor to the upper face of the collection-well plate 24or to the lower face of the drip-director plate 14. Such attachment maybe made by any suitable means, e.g., using fasteners, welds and/or oneor more adhesives, such as tapes, gums, cements, pastes, or glues.Instead of attaching the aerosol guard to a plate, the aerosol guard maysimply be sandwiched between the confronting surfaces of the plates andmaintained in place, for example, by frictional and/or compressiveforces.

The aerosol guard may be formed as a single sheet, e.g., about 0.10″ to0.15″ thick, or, alternatively, it may be formed of two or more sheets,e.g., each about 0.060″ to 0.065″ thick, arranged in layers. In onepreferred embodiment, a single-layer aerosol guard made of a poroushydrophilic polymer having compliant characteristics, such as ethylvinyl acetate (EVA) or the like, is attached to the lower face of thedrip-director plate using a pressure sensitive adhesive. Anotherembodiment contemplates a multi-layered construction, including: (i) aconformant layer comprising a foam pad, about 0.062″ thick and having apressure sensitive adhesive on both faces, and (ii) a porous, UHMW(ultra-high molecular weight) polymer layer, about 0.062″ thick, that ispermeable to air but substantially impermeable to aerosols. In thislatter embodiment, the conformant layer is attached to the lower face ofthe drip-director plate and then the UHMW polymer layer is attached tothe conformant layer.

Other materials (i.e., hydrophobic, non-polymeric, etc.) may be used informing the compliant aerosol guard of the present invention, providedonly that the material(s) effectively limits the passage of aerosols,while permitting the drawing of a vacuum therethrough.

In another embodiment, the means for avoiding cross-contamination due tothe well-to-well movement of aerosols includes vents or apertures 28extending through the surface of collection plate 24. In one preferredembodiment, at least one such aperture is disposed near each collectionwell. It should be appreciated that a reduced pressure applied frombelow the plate will extend through the apertures to the microfiltrationwells.

Any number and spatial configuration of apertures may be utilized,provided only that the region between each drip-director outlet andcorresponding collection well is disposed in fluid communication (i.e.,permissive of a vacuum) with the region below the collection plate alonga pathway that does not pass over the openings of neighboring wells. Forexample, an aperture may be provided centrally within a group of fourwells, with the wells being disposed about the corners of aquadrilateral. By providing 24 of such 4-well groupings, each well of astandard 96-well arrangement could be provided with a vent or apertureadjacent thereto. Alternatively, the number of apertures may equal orexceed the number of collection wells, with each well having one or moreclosely associated apertures proximate thereto. For example, a 96-wellcollection plate could be provided with at least 96 apertures arrangedso that each well has at least one closely-associated aperture. In thisregard, the apertures may be laid out, for example, in a 12×8, or 13×9,regular rectangular array.

As previously noted, apertures 28 permit fluid communication between theregions above and below the collection-well plate 24. Upon evacuatinglower vacuum chamber 29, a vacuum will be established reaching from exitport 51 to the region between each microfiltration well and acorresponding collection well. Particularly, the vacuum will pull alongflow pathways extending from each microfiltration well 18 into theinterface region between the confronting surfaces of drip-director plate14 and collection-well plate 24. The vacuum flow pathways then willcross downward through the collection plate's surface 25, by way ofrespective vents 28, to the open reservoir of chamber 29. Here, thevacuum flow pathways will extend along the lower chamber until reachingexit port 51. Large, blackened arrows illustrate exemplary vacuum flowpathways in FIG. 3. Advantageously, aerosols and filtrate residues thatbecome entrained in the vacuum flow are largely directed away from eachcollection well area and out of the system without passing overneighboring collection wells. Also, it should be appreciated that thevacuum pathways are directed in such a manner as to encourage a flowthat is largely downward and laminar in nature. Cross-flow, and thusturbulence, is greatly minimized compared to most conventionalarrangements.

The illustrated embodiments show a cross-flow restrictor 30 used incombination with a vented collection-well plate 24, as just described.Notably, the cross-flow restrictor 30 covers the apertures 28, so that avacuum pathway extending from the region between each microfiltrationwell 18 and corresponding collection well 26 to the region below thecollection-well plate 24, via a nearby aperture 28, must pass throughthe cross-flow restrictor 30. Since the cross-flow restrictor 30 allowsa vacuum to be drawn therethrough, but discourages the passage ofaerosols, filtrate-associated aerosols are substantially separated(i.e., filtered out by the cross-flow restrictor) from the drawn vacuumand, thus, the potential for well-to-well movement of aerosols over thecollection plate's surface 25 is even further reduced.

Instead of utilizing a unitary cross-flow restrictor for a plurality ofdrip directors and collection wells (e.g., a sheet having a plurality ofcircular perforations extending therethrough), as described above andshown in the accompanying drawings, an alternative embodimentcontemplates a plurality of individual collar or skirt-like cross-flowrestrictors. In horizontal cross-section, such individual cross-flowrestrictors can be of any suitable shape, e.g., annular, elliptical,oblong, etc. In one embodiment, each individual cross-flow restrictorco-axially and laterally surrounds the region between one drip directorand a corresponding collection well. Such cross-flow restrictors can beformed of a substantially rigid material, e.g., like that of thedrip-director plate, or they can be formed of a compliant, poroushydrophilic material, e.g., a polymer such as ethyl vinyl acetate (EVA)or the like. In one embodiment, a plurality of substantially rigid,annular or elliptical cross-flow restrictors are integrally molded withone of the trays, e.g., depending from the lower surface of thedrip-director plate and extending down toward the collection-well plate,about respective drip directors. Further, each such rigid cross-flowrestrictor is configured to allow a vacuum drawn from beneath acollection plate, situated under the drip-director plate, to extend tothe region proximate the encircled drip director. In this regard, eachcross-flow restrictor can be configured to encompass, in addition to acorresponding collection well, an adjacent aperture leading to theregion below the collection plate. That is, the cross-flow restrictorcan extend around both a corresponding collection well and an adjacentaperture. In an alternative embodiment, the cross-flow restrictorextends only around its corresponding collection well. That is, thecross-flow restrictor does not additionally encompass an adjacentaperture. Rather, in this embodiment, a small through-hole formed in thecross-flow restrictor, proximate the aperture, permits fluidcommunication between the aperture and the region proximate the dripdirector. It should be appreciated that, like the previously-describedsheet-like cross-flow restrictor 30, the individual cross-flowrestrictors shield against filtrate spattering and undesirable lateralmovement of aerosols across the upper surface of the collection-wellplate that can result in cross-contamination.

As previously mentioned, it is noteworthy that the vacuum flow pathwaysestablished between the regions above and below the collection-wellplate, in all of the embodiments described herein, are routed in amanner that encourages a largely laminar and downward flow (includingany entrained gases and/or aerosols). Compared to most conventionalarrangements, horizontal flow over the upper surface of thecollection-well plate is greatly minimized. Not only is this the case inthe regions proximate the microfiltration and collection wells, but itis also the case for the peripheral-edge regions of the plates. In thisregard, and with particular reference to the embodiment of FIG. 3, thecontact between the inwardly extending flap 44 a of stepped gasket 44and the top of ridge 48 of the collection-well plate 24 is such thatairflow therebetween is obstructed or baffled. Thus, upon evacuating thelower vacuum chamber 29, gases located above the stepped gasket 44, inthe region denoted by arrow 46, will be drawn into the lower vacuumchamber via vent 28. Gases in the space under the lower surface ofstepped gasket 44, denoted generally by the arrow 47, on the other hand,will be drawn into the lower vacuum chamber via a gap 49 providedbetween the collection-well plate and the surface 50 about vacuumchamber 29. By limiting the extent of horizontal airflow across thecollection-well plate in this way, turbulence resulting from cross flowalong the periphery of the arrangement is minimized.

An additional means for avoiding cross-contamination due to well-to-wellmovement of aerosols, as well as filtrate splattering, relates to thepositioning of each drip director's lower opening relative to the upperrim, or lip, of a corresponding collection well. According to thisfeature, the outlet port 16 c of each drip director 16 extendsdownwardly from the drip-director plate 14 so as to enter into acorresponding collection well 26. In this regard, the lower portion ofeach drip director 16 has a diameter that enables it to register withthe open top of a corresponding collection well 26 in the collectionplate 24. As shown in the embodiment of FIG. 3, the outlet port 16 c ofeach drip director 16 is situated below the upper rim or lip of acorresponding collection well 26. By placing the outlet port 16 c at aregion that is laterally surrounded by the inner sidewalls of thecollection well 26, much of the aerosol generated during filtration willimpact upon the collection-well walls, as opposed moving laterally overtoward a neighboring collection well. As an additional advantage, suchplacement of the drip-director outlets helps to reduce filtratesplattering.

In a related aspect, the present invention provides a method foravoiding cross-contamination due to well-to-well movement of aerosols ina multi-well microfiltration system. According to one embodiment, themethod includes the steps of:

(i) providing an array of microfiltration wells (containing fluidsamples) over a collection-well tray supporting a corresponding array ofcollection wells;

(ii) drawing a vacuum along flow pathways extending (a) from eachmicrofiltration well (b) downward through a plane defined by an uppersurface of the collection tray at a point at, or adjacent, acorresponding collection well (c) to a region beneath the collectiontray, thereby causing a filtrate to flow from each microfiltration welland into corresponding collection wells; and

(iii) obstructing aerosols formed from the filtrate at any onemicrofiltration well from moving across the upper surface of thecollection tray to a non-corresponding collection well, thereby limitingcross-contamination.

It should be appreciated that the apparatus described above isparticularly well suited for carrying out this method. For example, avacuum chamber, such as lower chamber 29 shown in FIG. 3, may beconnected to a low pressure source, such as a vacuum pump (not shown),for establishing a pressure differential across filter elements 8 a, 8 bdisposed in microfiltration wells 18. The reduced pressure, then, willcause filtrate to emanate from drip directors 16. Aerosol guard 30provides a means to limit filtrate-associated aerosols formed from thefiltrate at any one microfiltration well 18 from moving across the uppersurface 25 of collection-well plate 24 to a neighboring collection well.Apertures 28, extending through the surface 25 of collection plate 24,permit the vacuum to extend between each microfiltration well and theregion below the collection-well plate 24 without having to pass overthe openings of neighboring collection wells.

When evacuating the lower chamber, it is advantageous to slowly change(ramp) the pressure to a desired value, combined with the utilization ofvery low pressures (e.g., less than about 2 psi, and preferably lessthan about 1 psi), in to further reduce the potential forcross-contamination, as by aerosols. For example, in going from ambientpressure to a value within the range of about 0.75 to about 2 psi, aramp period of about 2-3 seconds is employed.

Another aspect of the present invention pertains to a multi-wellmicrofiltration arrangement that provides for the flow of filtrate fromeach well, while avoiding crosscontamination due to pendent drops whichmay adhere to the drip directors of the various microfiltration wells.As previously mentioned, such pendent drops can fall into neighboringcollection wells when moving the drip-director plate over thecollection-well plate.

According to one embodiment, a microfiltration well is evacuated in thedirection of its upper opening, thereby pulling any pendent drops offluid hanging from its drip director back up into the well. Toaccomplish the evacuation, a pressure control source, e.g., a vacuumpump, in communication with an upper region of the mini-column isoperable to evacuate the mini-column in the direction extending from thedrip director to the upper opening.

Another embodiment provides for “touching off” the tips of the dripdirectors to remove pendent drops of filtrate that might hang off of thedrip directors. In this regard, the drip director outlets of all themicrofiltration wells are simultaneously brought into contact with theinner sidewalls of corresponding collection wells.

Means are provided for effecting relative motion between thedrip-director plate and the collection-well plate for simultaneouslymoving the discharge conduits into and out of contact with inner wallsof respective collection wells. In one embodiment, such means areoperable to shift the collection-well plate along a plane substantiallyorthogonal to the longitudinal axes of the microfiltration wells, whilethe microfiltration wells themselves are maintained in a substantiallyfixed position. In another embodiment, the means for effecting relativemotion are operable to shift the microfiltration wells along a planesubstantially orthogonal to the longitudinal axes of the collectionwells, while the collection wells are maintained in a substantiallyfixed position.

An exemplary arrangement for effecting relative motion is depicted inFIGS. 7 through 10. With initial reference to FIGS. 7 and 8, an L-shapedcarriage, as denoted by the reference numeral 60, is provided with acentral opening 62 configured to receive and support a multi-wellmicrofiltration assembly, indicated generally as 6, from above. Belowcarriage 60, a collection plate 24 having an array of collection wells26 is supported in a lower vacuum chamber (not shown).

Carriage 60 is mounted on a pair of parallel longitudinal carrier railsfor reciprocal linear motion along a first, substantially horizontal,axis. In the illustrated embodiment, one of the carrier rails is alinear bearing rail, denoted as 64, which supports the carriage 60 viaan interposed linear bearing member 65 attached to the lower surface ofthe carriage 60 toward one lateral edge. The other carrier rail is aU-shaped bearing guide, denoted as 66, that receives a bearing wheel 68,extending laterally outward from the other edge of the carriage 60, inan elongated track or slot 66 a.

Carriage 60 is moved along the rails 64, 66 by a belt assembly comprisedof a flexible belt 70 having its ends attached at each longitudinal endof a U-shaped bracket 74 forming a part of a spring-loadedmotion-control mechanism 72, described more fully below. Belt 70 ispassed around a driven 76 roller and an idler roller 78, disposedproximate longitudinally opposing ends of the carrier rail arrangement.To prevent against slippage, the belt may be provided with teeth 70 aadapted for mating engagement with complementary sets of teeth 76 a, 78a on the rollers.

Driven roller 76 is in mechanical communication with a motor, such as82, through a power train assembly, as indicated generally by thereference numeral 84. When motor 82 is energized, belt 70 will move,causing carriage 60 to slide along the carrier rails 64, 66, with thedirection of movement depending on the rotation of the drive shaft 86extending from motor 82. Motor 82 may be of any suitable, known type,e.g., a stepper motor, servo motor, or similar device.

One preferred embodiment of the present invention contemplates the useis of a stepper motor to move belt. By way of background, a steppermotor is a specialized type of motor that moves in individual steps.Unlike servo motors, the position of a stepper can be determined withoutthe need for expensive encoders to check its position. Stepper motorsare much more cost-effective than servo systems due to their simplifiedcontrol and drive circuitry. There are no brushes to replace in astepper motor, reducing the frequency for maintenance. Owing to theirease of use and relatively low cost, steppers are often preferred overservo motors for many modern computerized motion control systems.

According to this embodiment of the invention, a control system isprovided to operate the stepper motor in a desired fashion. For example,a microcontroller, such as a Motorola 68332, may be utilized to controlthe motor using conventional techniques.

As previously noted, stepping the motor 82 causes belt 70 to move aroundrollers 76, 78, with the direction of movement dependent upon thedirection of rotation of the motor's shaft 86. Movement of belt 70, inturn, causes carriage 60 to slide along guide rails 64, 66, therebyshifting the drip director array 16 laterally with the respect to thecollection well array 26. If the drip directors 16 are positioned sothat they extend into respective collection wells 26, sufficientstepping in a given direction will cause the drip directors 16 to engagethe upper, inner surfaces of the collection wells 26, as shown in thesectional views of FIGS. 9(A)-9(C). In this way, pendent drops offiltrate hanging from the drip directors 16 are “touched off” to theinner surfaces of respective collection wells 26. Similarly, uponreversing the stepping direction, the drip directors 16 can be moved toengage the upper, inner surfaces on the opposing side of the collectionwells 26 to further ensure effective touching off of pendent drops.

As previously mentioned, alternative embodiments of the inventioncontemplate the use of a servo motor to move the belt. In one suchembodiment, a means for providing positional feedback, such as anencoder (not shown), is provided in order to track the position of theservo motor.

Carriage additionally supports means for moving and positioning themicrofiltration arrangement 6 along a second, generally vertical, axis.With particular reference to the embodiment of FIG. 7, avertical-positioning mechanism is disposed on the upper surface ofcarriage along each lateral side of the microfiltration arrangement.Each vertical-positioning mechanism includes (i) lift springs, such as92, that provide a continuous, upwardly-directed force tending to raisethe microfiltration arrangement 6 to an elevated position whereat thedrip directors 16 fully clear the upper lips of the collection wells 26,and (ii) fluid cylinders, such as 94, that are operable to lower themicrofiltration arrangement 6, against the force of the lift springs 92,to a seated position whereat each drip director 16 extends into theupper region of a respective collection well 26. At its fully seated(lowered) position, the microfiltration arrangement 6 forms a seal withthe lower vacuum chamber (not shown).

Both the springs 92 and the fluid cylinders 94 engage, at their upperends, handles, denoted as 96, that extend upwardly and outwardly fromeach lateral side of the microfiltration arrangement's supporting frame38. In one embodiment, the spring/cylinder arrangements are operable tohold the microfiltration arrangement at any one of three positions: (i)an up or travel position, (ii) a touch-off position, and (iii) a down orseal position.

The touch-off operation may be carried out with the microfiltrationarrangement 6 disposed at any position along the second (vertical) axis,provided only that the drip directors 16 extend at least partially downinto the collection wells 26. In one embodiment, touching off of thedrip directors 16 to the inner sidewalls of the collection wells 26 iseffected with the microfiltration arrangement 6 slightly raised aboveits fully seated position so that the lowermost regions of the dripdirectors 16, proximate their outlets 16 c, will abut the inner surfacesof the collection wells 26.

The region of each drip director 16 proximate its outlet may be shaped,e.g., angled or chamfered about its lower circumference, to promote thelocalization of any pendent drops of filtrate to certain regions of thedrip director 16 and to optimize contact between such regions with theinner sidewall of a corresponding collection well 26 during touch off.Similarly, the upper region of each collection well 26 may also beshaped, e.g., in a manner complementary to (i.e., matching) a shapeddrip director 16, so that adequate contact is made between theseelements during touch off for substantially ridding the drip director 16of any pendent drops of filtrate. In one preferred embodiment, as can beseen in FIGS. 9A-C, the upper, region of each collection well is formedwith an outwardly angled inner sidewall that matches an inwardly angledouter surface along the lower region of a corresponding drip director,thereby providing a substantial abutting surface between these elementsduring a touch-off operation.

As previously described, the discrete quantity of angular rotationimparted to shaft 86 each time stepper motor 82 is stepped is ultimatelytranslated into a given length of linear movement by bracket 74. Forexample, stepping the motor 82 once may cause bracket 74 to move ¼″ in aparticular direction. It should be appreciated that the minimum numberof steps required of stepper motor 82 to effect a touch off may causethe drip directors 16 to move farther than what is necessary. That is,the drip directors 16 might be moved into engagement with the innerwalls of the collection wells 26, with continued pressure to move beyondthe inner walls. As described next, such linear overshoot can beadvantageous, as it can assist in the removal of pendent drops. Itshould be appreciated that it is desirable to move the drip directors toa suitable position against the collection-well sidewalls (e.g., in firmabutment with the sidewalls) in order to effectively encourage theremoval of pendent drops. By providing a suitable amount of linearovershoot into the sideward movement of the drip directors, suchpositioning can be ensured (i.e., the drip directors will not fall shortof the sidewalls), notwithstanding various minor positional inaccuraciesinherent in the arrangement. Thus, by providing for a reasonable amountof linear overshoot, the sidewalls themselves determine the finalposition of the drip directors. It is also desirable to keep the torquerelatively low, thereby preventing clogging of the motor. Further, it isdesirable to absorb or compensate for some of the linear overshoot toavoid overstressing the drip directors 16 and/or the collection wells26.

In these regards, one embodiment of the invention contemplates the useof a spring-loaded motion control mechanism 72 in the mechanical linkagesystem between the motor 82 and the carriage 60. The motion-controlmechanism 72 ensures accurate positioning of the drip directors inabutment with the sidewalls, while absorbing excess linear motion beyondthe amount required to shift the drip directors 16 into contact with theinner sidewalls of the collection wells 26. As an additional advantage,the motion-control mechanism 72 provides a damping resistance to slidingmovement of the carriage 60 along the rails 64, 66.

In one embodiment, the motion-control mechanism includes a springdisposed such that movement of the carriage in either direction alongthe first axis will put the spring under compression. With particularreference to the partially schematic top plan views of FIGS. 10(A)-(C),the U-shaped bracket 74 that forms a part of the belt assembly isrigidly connected to a housing 101 containing large and small bores,respectively indicated generally as 102 and 108. Bore 102 has alarge-diameter portion 102 a and a small-diameter portion 102 b,separated by a radial step 102 c. A stepped-diameter shaft, indicatedgenerally as 104, having a large-diameter portion 104 a and asmall-diameter portion 104 b, separated by a radial step 104 c, passesthrough bore 102 and rigidly attaches, at its large-diameter end, to anextended-arm portion 60 a of the L-shaped carriage 60. A guide pin 106,which assists in maintaining the substantially horizontal orientation ofcarriage 60, rigidly attaches to the extended arm portion 60 a ofcarriage 60 at one end and is received in small bore 108 at its otherend. Inside the large-diameter portion 102 a of bore 102, a spring 110concentrically mounts the small-diameter portion 104 b of shaft 104between a pair of spaced washers, denoted as 112 and 116. The twowashers 112, 116 are concentrically mounted for sliding movement alongthe small-diameter portion 104 b of stepped shaft 104. Spring 110 urgesthe two washers 112, 116 toward opposite, extreme ends of thesmall-diameter portion 104 b of shaft 104. A fixed-position washer 114is seated within a circumferential groove (not shown) formed in thesmall-diameter portion 104 b of shaft 104 near its free end.

When belt 70 moves U-shaped bracket 74 in the direction indicated by thearrow “A,” in FIG. 10B, bore 102 slides along shaft 104 in a directiontoward the extended arm 60 a of carriage 60. As a result, an annular lip120 that extends radially inward at the end of bore 102 acts against anannular, peripheral region of washer 112, causing the washer 112 toslide along the small-diameter portion 104 b of stepped shaft 104,thereby compressing spring 110. When the compression force overcomes thepre-loaded retaining force, carriage 60 will then shift in the samedirection (direction “A”).

When belt 70 moves U-shaped bracket 74 in the direction indicated by thearrow “B,” in FIG. 10C, bore 102 slides along shaft 104 in a directionaway from the extended arm 60 a of carriage 60. As a result, the radialstep 102 c of bore 102 acts against an annular, peripheral region ofwasher 116, causing the washer 116 to slide along the small-diameterportion 104 b of stepped shaft 104, thereby compressing spring 110. Whenthe compression force overcomes the pre-loaded retaining force, carriage60 will then shift in the same direction (i.e., direction “B”).

In one embodiment, spring 110 provides a pre-load force of about 1pound. Thus, the force provided by the stepper motor 82 will not beeffective to move the carriage 60 until the threshold of about 1 poundis overcome. Advantageously, the arrangement provides (i) aconstant-hold mode at the center, or neutral, position, and (ii) aconstant-force mode for effecting touch off. The spring 110 providescompliance in the system, e.g., allowing touch off to start at 1 poundand end at 1.2 pounds.

With reference to the apparatus as described above, one preferredembodiment of the present invention contemplates the following steps:

(i) microfiltration arrangement 6 is loaded onto carriage 60 and clampedin place;

(ii) carriage 60 is centered over a lower vacuum chamber 29;

(iii) microfiltration arrangement 6 is lowered to its seated position(e.g., by retracting fluid cylinders 94) and sealed over the lowervacuum chamber 29;

(iv) a robot (not shown) lowers upper vacuum chamber 20 against the topof microfiltration arrangement 6 and, optionally, applies a downwardforce, e.g., about 5 pounds, to the stacked arrangement;

(v) lower vacuum chamber 29 is evacuated (e.g., at about 0.5-3 psi) toeffect elution/purification;

(vi) carriage 60 is raised slightly from its fully seated position to atouch-off height whereat only the lowermost regions of the dripdirectors 16 extend below the upper lips of the collection wells 26;

(vii) motor 82 is stepped in a forward direction to touch off the dripdirectors 16 to a sidewall of the collection wells 26;

(viii) motor 82 is stepped in a reverse direction to touch off the dripdirectors 16 to the opposing inner sidewall of the collection wells 26;

(ix) forward and reverse stepping of motor 82 is repeated to performeach of the touch-off steps once more;

(x) carriage 60 is re-centered over lower vacuum chamber 29;

(xi) microfiltration arrangement 6 is lowered to its seated position andsealed over lower vacuum chamber 29;

(xii) optionally, the robot can apply a downward force, e.g., about 5pounds, to the stacked arrangement;

(xiii) upper vacuum chamber 20 is evacuated to effect a pull-back ofpendent drops (e.g., at about 0.1-0.3 psi);

(xiv) microfiltration arrangement 6 is raised to its fully elevatedposition so that the drip directors 16 fully clear the collection wells26; then

(xv) carriage 60 is moved to next station.

FIG. 16 shows an automated high-throughput sample preparationworkstation 202, including, for example, a microfiltration apparatus,cross-contamination control arrangements, as well as collection-wellcovering and heat-sealing assemblies (described below), and associatedcomponents and reagents, in accordance with the teachings of the presentinvention. As illustrated, several collection trays can be provided inadjacent vacuum chambers arranged in a side-by-side fashion near one endof the workstation. For example, a closed-bottom collection tray, suchas tray 24, can sit in each of the two endmost vacuum chambers, whileopen-bottom collection trays can sit in the two center vacuum chambers.Carriage 60 can then carry a microfiltration arrangement 6 successivelyfrom one vacuum chamber to the next. For instance, an initial collectionof filtrate can take place at the vacuum chamber holding closed-bottomcollection plate 24 near the front of the workstation. Then successivewashings can be carried out at each of the two center vacuum chamberswhereat open-bottom collection plates are placed. Next, a finalcollection of filtrate can take place at the vacuum chamber near therear of the workstation, whereat another closed-bottom collection trayis located.

With regard to spatial orientation, it should be noted at this pointthat the various components (e.g., upper chamber, mini-column plate,filter element, drip-director plate, frame, cross-flow restrictor,collection-well plate, and lower chamber) are illustrated and describedherein as being stacked in vertical relationship, with the upper vacuumchamber being the topmost component. Further, each microfiltration wellis described as having a central axis disposed in a substantiallyvertical fashion, with a flow pathway extending downwardly through thewell. It should be noted, however, that these orientations have beenadopted merely for convenience in setting forth the detaileddescription, and to facilitate an understanding of the invention. Inpractice, the invention contemplates that the components and wells maybe disposed in any orientation.

In another of its aspects, the present invention provides for thecovering and sealing of multi-well trays containing fluid samples.

In one embodiment, shown in FIGS. 11 through 14, a cover member,indicated generally by the reference numeral 150, includes an uppershell portion, denoted generally as 154, supporting a sealing layer orundersurface, indicated generally as 156 (FIG. 11), on its lower face.Upper shell portion 154 is comprised of a substantially planar expanse158 (FIGS. 12 and 13) and a depending circumferential sidewall 160laterally surrounding undersurface 156. Along the length and widthdimensions, undersurface 156 is configured with generally the samegeometry as the upper surface of multi-well plate 24, permitting it tocover the entire array of well openings 26 a.

As best seen in FIG. 11, undersurface includes a plurality of individualnodules, such as 166, arranged in a rectangular array corresponding tothe array of wells 26 of collection tray 24. Each of the nodules 166preferably has a downwardly convex, e.g., dome-shaped, lower portion,though other shapes may be used. The nodules 166 are made of aresiliently flexible material held in a predetermined, spacedrelationship from each other by a web or sheet 168. Web 168 may beintegrally formed with nodules 166, as shown, or it may be formedseparately, with the nodules molded or adhesively attached to the web atappropriate locations.

Cover 150 is preferably comprised of a substantially rigid materialthat, when pressed down at opposing peripheral edge regions againstcorresponding regions of ridge 48 along the periphery of multi-wellplate 24, can maintain an annular region of each nodule 166 in pressingengagement with an upper lip 26 b of a respective well 26. To evenlydistribute the downward force across undersurface 156, integral beams,such as 172 and 174, can extend laterally and/or longitudinally acrossthe top surface of upper shell portion 154, providing increasedrigidity.

Undersurface 156 is formed of a resiliently deformable material that,when compressed over openings 26 a, is capable of forming a seal.Suitable materials for forming undersurface 156 include, for example,synthetic rubber-like polymers such as silicone, sodium polysulfide,polychloroprene (neoprene), butadiene-styrene copolymers, and the like.Upper shell portion 154 is formed of a substantially rigid material suchas nylon, polycarbonate, polypropylene, and the like.

In one preferred embodiment, the cover of the invention is made by aninjection co-molding process wherein an upper shell portion is firstmolded, and then a sealing undersurface is injection molded to the shellportion. A preferred nylon material useful for forming the upper shellis available commercially as ZYTEL® grade 101 (DuPont Co., Wilmington,Del.). To avoid heat-induced damage to the molded nylon shell portion,preferred silicone materials have relatively low injection and curingtemperatures (e.g., less than about 180° C). One particularly preferredsilicone material useful for co-molding the undersurface is availablecommercially as COMPU LSR 2630 clear (Bayer AG, Germany).

To secure undersurface 156 to upper shell portion 154, a series of holes(not shown) are formed through the shell's planar expanse 158. Uponinjecting a liquid silicone from the bottom side of the upper shellportion 154, the silicone penetrates the holes and forms nodules, suchas 180, having a greater diameter than that of the holes, adjacent thetopside of the upper shell portion 154. Upon curing, the siliconecontracts slightly, pulling the nodules 180 and the expansiveundersurface 156 toward one another. In this way, a snug attachment iseffected at several locations holding the undersurface 156 against thelower face of the upper shell portion 154.

Cover 150 is secured to multi-well tray 24 by a releasable attachmentmeans. In the embodiment of FIGS. 11-14, the attachment means includes aplurality of integrally formed, resiliently deflectable arms, such as184, depending from opposing lateral sides of upper shell portion 154.At an end distal from upper shell portion 154, each arm 184 is providedwith a catch or hook 186 adapted to hold on to a circumferentialsidewall 24 a formed about multi-well plate 24. As best seen in FIGS. 11and 14, each catch 186 is substantially formed in the shape of ahalf-arrow, having (i) a downwardly and outwardly angled cam surface 186a, and (ii) an upper shoulder or stop portion 186 b. Upon moving thecover 150 toward a seated position over the well openings 26 a, the camsurface 186 a of each catch 186 slides down over the circumferentialsidewall 24 a of collection plate 24, thereby deflecting arms 184laterally outward. Once the shoulder 186 b of each catch 186 clears thelower edge 24 b of circumferential sidewall 24 a, arms 184 snap inward,locking the cover 154 in a closed position, as shown in FIG. 13.

To release the snap-locked cover 154 from multi-well tray 24, arms 184can be pulled outwardly, away from circumferential sidewall 24 a, sothat each shoulder 186 b clears lower edge 24 b. Cover 154 can then beseparated from the tray 24 to reveal the well openings 26 a.

In an alternative embodiment, shown in FIG. 15, the releasableattachment means includes a plurality of nubs or protrusions 192 havingresiliently deformable terminal bulbs 192 a depending at various pointsalong the periphery of the lower surface of cover 154. In thisembodiment, nubs 192 are receivable within complementary bores 194formed along corresponding regions of the upper surface of multi-wellplate 24. Frictional engagement of each bulb 192 a with an innersidewall of a respective bore 194 holds the cover 154 in place over themulti-well plate 24.

In one preferred embodiment, best seen in FIG. 12, structure is providedalong the top of upper shell portion 154 that facilitates automatedhandling using a robotic fluid-handling apparatus. An exemplary roboticsystem is available commercially under the tradename TECAN® RSP (TecanAG; Hombrechtikon, Switzerland). In the illustrated arrangement, therobotic system, denoted generally as 198, includes four elongatedaspiration and injection fingers, denoted as 1-4, mounted on a roboticarm 200 at respective points generally defining a line. Arm 200 cantranslocate fingers in the x/y direction along a generally horizontalplane, throughout which the longitudinal axes of fingers 1-4 aremaintained in fixed, spaced relation to each other. The longitudinalaxes of fingers 1-4 are evenly spaced from about 9 mm to about 36 mm,and preferably about 18 mm, apart from one another. Each of the fingers1-4 can be separately translocated in the z direction along arespective, generally vertical axis. Movement of arm 200 and fingers 1-4is preferably carried out under the control of a programmed computer(not shown) by known techniques.

The TECAN® RSP can be used, in a known manner, to transfer fluids to andfrom various chambers, e.g., wells of a microplate 24, vials 206,troughs 208 and the like, disposed on a working surface, such as theworktable 202 shown in FIG. 16. Other known uses for the TECAN® RSPinclude, for example, reagent addition, dilution, and mixing.

As previously mentioned, the present invention provides structure alongthe top of upper shell portion 154 that facilitates automated handling.Advantageously, such structure expands the capability of the roboticsystem 198 beyond conventional fluid-handling tasks to include noveltasks such as picking up covers, placing covers over multi-well plates,and securing the covers to the plates, as described below. As shown inthe embodiment of FIG. 12, such structure can include longitudinallyslotted, resiliently expandable sleeve-like members, such as 211 and214, each adapted to receive the tip region of one of the fingers 1-4such that the finger becomes wedged therein. Such structure furtherincludes a plurality of landing seats, e.g., 221-224 and 212-213,defined by rimmed depressions or bores having a diameter wider than thatof fingertips (1 a-4 a), providing strategic regions whereat the fingerscan abut the upper surface of the cover with a reduced risk of slippage.

Generally, frictional engagement of each sleeve 211, 214 with one offingertips 1 a-4 a permits the robot to pick up, carry and/or placecover 150, as desired. Once suitably placed, the cover can be releasedby extending one or more free fingers against corresponding landingseats on the upper surface of the cover, while retracting the wedgedfingertips. In an exemplary operation, fingers 1, 4 are extendeddownward in the z direction toward a cover 150 disposed, for example, ona surface of a workstation 202 so that fingertips 1 a, 4 a enter andbecome wedged within respective expandable sleeves 211, 214. Fingers 1,4 are then partially retracted in the z direction, in unison, to liftcover 150 above the working surface. Next, the lifted cover 150 istranslocated by moving arm 200 along the x/y direction to another areaof the working surface. Fingers 1, 4 are then extended downward, inunison, in the z direction to lower cover 150 onto a multi-well tray 24containing, for example, a plurality of separately collected fluidsamples. Cover 150 is released from the robot 198 by extending freefingers 2, 3 downward against landing seats 212, 213 on the uppersurface of cover 150, and retracting fingers 1, 4 from sleeves 211, 214.Finally, all of the fingers (14) are raised toward arm 200 to a fullyretracted position.

Employing a releasable attachment means, cover 150, so placed, can besnap-locked to the multi-well tray 24 by applying a downward force fromabove. In an exemplary operation, fingers 1, 4 are extended downward inthe z direction to abut landing seats 221 and 223, respectively, on thetop of cover 150. The downward motion of finger 1 is continued againstlanding seat 221 so that the locking arm 184 thereunder is moved intosnapping engagement with circumferential sidewall 24 a of multi-welltray 24. In the meanwhile, finger 4 is held substantially motionlessadjacent landing seat 223 in order to oppose any tendency of the coverto flip up. An appropriate downward force is then applied at landingseats over the remaining arms until all of the arms are snap-locked tothe multi-well tray.

While it should be understood that the covers described herein can beemployed in a wide variety of situations, they are particularly usefulfor protecting a plurality of fluid samples separately contained in anarray of closed-bottom collection wells against evaporation and/orcross-contamination during long-term storage. For example, fluidfiltrate (e.g., containing purified or concentrated nucleic acids, suchas RNA or DNA) can be collected using the microfiltration apparatus ofFIGS. 1 to 3. The collection wells can then be sealed by snap-lockingthe cover 150 of FIGS. 11-14 thereto, using a TECAN® RSP fluid-handlingrobot as just described. The covered multi-well tray can then be placedin a freezer for storage. The covers can be reused multiple times, ifdesired.

By utilizing a single robotic fluid-handling arm to carry out a varietyof tasks at the workstation, valuable working space is conserved.Moreover, equipment and programming expenses are avoided by obviatingthe need for additional robotic devices, e.g., grippers, for picking up,moving, placing and securing covers.

The present invention also provides for the sealing of multi-well trayswith heat-sealable covers. Generally, the heat-sealing apparatus of theinvention includes a pick-and-place assembly adapted to lift anindividual, pre-cut, heat-sealable sheet or film from a bin and placethe sheet over a plurality of well openings of a multi-well tray. Aheatable platen is provided for engaging the sheet, so placed, and heatsealing the sheet to the upper surface of the multi-well plate, therebyforming a seal over each well. Advantageously, the operation is carriedout in an automated fashion.

According to one embodiment, shown in FIGS. 16-24, a first,substantially planar work surface, generally denoted as 302, ispositioned over a cooler, indicated generally by the reference numeral306. A plurality of rectangular cavities, such as cavities 310, areformed through the work surface, each adapted to support a multi-welltray therein, such as tray 324, with the lower side of the tray disposedin communication with the temperature-controlled environment of cooler306. In a preferred embodiment, four such cavities are formed throughwork surface 302. When a multi-well tray is properly positioned in oneof cavities 310, an outer circumferential edge or lip of the tray restson a region of the surface circumscribing the cavity, with the bottomregions of the wells extending below the surface into the cooler. Cooler306 is adapted to maintain the samples at a desired, reduced temperature(e.g., about 4° C. for fluid samples containing purified or concentratednucleic acids, such as RNA).

Means are provided to ensure proper placement (i.e., orientation) of amulti-well tray in a respective cavity. According to one embodiment, oneface of a triangular key feature, indicated as 322, is rigidly attachedto the work surface proximate a corner of each cavity 310. Further, onecorner of each multi-well tray is angled, as at 324 d, to sit on worksurface 302 closely adjacent the edge of key 322 facing the cavity 310,when the tray is properly (fully) seated. It should be appreciated thatonly the angled corner 324 d of tray 324 can sit on work surface 302adjacent key feature 322. If tray 324 is placed in the cavity in thewrong orientation, a non-angled comer of tray 324 (i.e., one other than324 d) will land on the upper face of key 322, thereby prohibitingproper (full) seating of tray 324 in manner that will be apparent to anoperator.

A second, substantially planar working surface, denoted as 332, ispositioned alongside first surface 302, such that the two surfaces aresubstantially coplanar. A bin or tray, indicated as 336, for holdingindividual sheets of heat-sealable covers is held in a frame, denotedgenerally as 338, affixed near one end of second working surface 332.Tray 336 is adapted to hold a plurality of heat-sealable sheets, denotedgenerally as 342, vertically stacked face-to-face, such that the topmostsheet is always presented for retrieval by a suction picker assembly,denoted as 394, at substantially the same, predetermined verticalheight. For example, tray 336 can rest on a spring-biased bed (notshown) adapted for vertical motion within frame 338.

Tray 336 is preferably formed as an integral unit using a conventionalthermoforming process. In one embodiment, tray 336 is formed of alightweight plastic material, such as PETG, or other suitablethermoplastic resinous material. As best seen in FIG. 18, tray 336 isformed with a bottom 336 a, four sidewalls 336 b, an outwardly extendingcircumferential lip 336 c, and an open top. As is well known in the artof thermoforming, it is generally necessary to provide a draft for thesidewalls of a thermoformed tray. Draft is the slight taper provided ina design of a thermoformed part that permits the part to be removed fromthe mold, after curing, without disturbing the part's walls. In thethermoformed tray of the present invention, the distance betweenopposing sidewalls of the tray slightly increases along the directionfrom the bottom of the tray to the top of the tray. For example, thesidewalls can be provided with a lift-out slope in the range of about1-10 degrees. In one embodiment, the lift-out slope is about 5 degrees.

As a consequence of the draft, the planar area bounded by the tray'ssidewalls 336 b, parallel to the tray's bottom 336 a, gets progressivelylarger moving along a direction from the bottom 336 a to the top of thetray. To prevent shifting of the sheets (not shown in FIG. 18) heldwithin the tray, particularly at the wider, upper regions thereof, ribs,such as 337, are provided along each sidewall 336 b. Ribs 337 areconfigured to provide a substantially straight surface or edge forcontinuously contacting a point, and preferably plural points, on eachperipheral side-edge of each sheet of a stack, throughout each sheet'srange of vertical motion. Thus, ribs 337 serve to guide each sheet as itis moved vertically through tray 336, and to ensure that each sheet ismaintained in a desired orientation within the tray 336. In theillustrated embodiment, ribs 337 are provided as integrally molded,opposing pairs extending along opposing sidewalls of the tray. The ribsrunning along each sidewall 336 b are sufficiently spaced apart so as toprevent the sheets held in the tray from becoming skewed. In thisembodiment, each rib 337 provides a vertically extending, elongated lineor surface that is substantially normal to a plane defined by the bottom336 a of tray 336. Due to the upwardly divergent nature of the tray'ssidewalls 336 b, the ribs 337 become slightly more pronounced (i.e.,they extend further outward from each sidewall's major surface) alongthe direction from the bottom 336 a of tray 336 to the top of tray 336.

A releasable attachment means is provided to prevent inadvertent removalof tray 336 from frame 338. In one embodiment, the attachment meansincludes resiliently deflectable arms, such as 350, extending upwardlyfrom opposing lateral sides of frame 338. Each arm 350 is rigidlyattached near its lower end, e.g., by way of fasteners 352, to a lowerregion of frame 338 proximate work surface 332. As best seen in FIGS.19(A)-19(B), the upper region of each arm 350 is provided with a catchor hook 356 adapted to hold on to the circumferential lip 336 c at thetop of tray 336 when the tray is disposed in a fully seated position.Each catch 350 is substantially formed in the shape of a half-arrow,having (i) an upwardly and outwardly angled cam surface 356 a, and (ii)a lower shoulder or stop portion 356 b. Upon moving tray 336 toward aseated position within frame 338, the cam surface 356 a of each catch356 slides over the outer peripheral edge of lip 336 c, therebydeflecting each arm 350 laterally outward. Once the lip passes belowshoulder 356 b, the arms snap inward, locking the tray in the frame, asshown in FIG. 17.

To release the snap-locked tray 336 from frame 338, arms 350 can be bentoutwardly, apart from one another, so that each shoulder 356 b clearsthe outer edge of lip 336 c. Tray 336 can then be lifted out of frame338.

An abutment, denoted as 362, is rigidly secured on the upper surface offrame 338 near each longitudinal end. Abutments 362 providesubstantially vertical, confronting surfaces that guide tray 336 as itis being placed in frame 338, and that maintain the tray in a desiredposition while it is seated.

Sheets 342 may be made of any substantially chemically inert materialthat can form a seal with the upper surface of a multi-well tray, orappropriate regions thereof (e.g., an upstanding rim or lip about theopening of each well), when applied with moderate heat (e.g., 90°14 170°C.) under moderate pressure (e.g., about 10-35 lbs.). For example,sheets 342 may be formed of a polymeric film, such as a polystyrene,polyester, polypropylene and/or polyethylene film. Suitable polymericsheets are available commercially, for example, from Polyfiltronics,Inc. (Rockland, Mass.) and Advanced Biotechnologies (Epsom, SurreyEngland UK). In one embodiment, each sheet is a substantially clearpolymeric film, about 0.05 millimeters thick, that permits opticalmeasurement of reactions taking place in the wells of tray 324. Forexample, the present invention contemplates real time fluorescence-basedmeasurements of nucleic acid amplification products (such as PCR) asdescribed, for example, in PCT Publication WO 95/30139 and U.S. patentapplication Ser. No. 08/235,411, each of which is expressly incorporatedherein by reference. Generally, an excitation beam is directed through asealing cover sheet into each of a plurality of fluorescent mixturesseparately contained in an array of reaction wells, wherein the beam hasappropriate energy to excite the fluorescent centers in each mixture.Measurement of the fluorescence intensity indicates, in real time, theprogress of each reaction. For purposes of permitting such real timemonitoring, each sheet in this embodiment is formed of a heat-sealablematerial that is transparent, or at least transparent at the excitationand measurement wavelength(s). A preferred heat-sealable sheet, in thisregard, is a co-laminate of polypropylene and polyethylene.

Often, heat-sealable films or sheets are obtained as a web in the formof a roll. Not surprisingly, such rolls are often bulky and heavy.Moreover, the equipment required to properly cut them into a desiredshape can be costly and space consuming, as well. Advantageously, theheat-sealable sheets provided by the present invention are pre-cut toappropriate dimensions, and stacked inside tray. As contemplated herein,a tray, such as 336, holding a stack of sheets 342 is packaged as apre-assembled unit, which is readily opened and inserted into frame 338.

A linear track 370, supporting a carriage assembly 376, is mountedlongitudinally across work surface 332, adjacent to cooler 306. Areversible drive is means is adapted to move carriage 376 back and forthalong track 370, as desired. In the illustrated embodiment, the drivemeans includes a nylon rail 382 having upwardly facing teeth 378 formedalong its top surface, from one end to the other. Teeth 378 are adaptedto mesh with a circular, extemally-toothed, motor-driven gear (notshown) disposed for rotation inside the carriage housing. A flexibleguide or conduit 386, as can be obtained commercially from KabelSchleppAmerica, Inc. (Milwaukee, Wis.), is disposed alongside rail 382 forcontaining various cables and wires (not shown) of the apparatus.

Carriage 376 supports a pick-and-place assembly, indicated generally bythe reference numeral 388, and a heatable platen assembly, denoted as412. Pick-and-place assembly 388 includes an elongated picker arm 390supported above the carriage via a rotatable mount 392 extending throughan upper surface of carriage 376. The rotatable mount can be, forexample, a driven shaft coupled to a reversible stepper motor (notshown) held within the carriage housing. Arm 390 is attached to drivenshaft 392 so as to rotate therewith. Arm 390 is adapted for movementalong a generally horizontal plane between a “home” postion (FIGS. 16and 23), a “pick-up” position (FIGS. 17 and 20), and a “drop-off”position (FIGS. 21 and 22).

The other end of arm 390 supports a suction picker assembly, indicatedgenerally by the reference numeral 394, adapted to pick up aheat-sealable sheet S from stack 342 held in tray 336, and to place thesheet over a multi-well tray held in one of the cavities at cooler 306.Suction picker assembly 394 includes four elongated guide rods, denotedas 396 a-396 d, each supported for reciprocal sliding movement within arespective linear bearing 398 a-398 d held in a bore (not shown)extending vertically through arm 390. Rods 396 a-396 d are secured, attheir lower ends, to the top of a plenum, denoted as 400.

Plenum 400 can be moved up and down to a desired vertical height by wayof a linear motion means. The linear motion means can be a steppermotor, such as 402, mounted on arm centrally of rods 396 a-396 d.Rotational motion of stepper motor 402 causes a lead screw 404, passingcentrally through motor is 402, to move up or down along itslongitudinal axis, dependent upon the direction of rotation. The lowerend of lead screw 404, in turn, is rotatably journaled to plenum 400.Thus, upon stepping motor 402, plenum 400 will move up or down withlinear movement of lead screw 404.

A plurality of suction legs, such as 406 a-406 d, depend from a lowerside of plenum 400. In the illustrated embodiment, one such leg isdisposed near each comer of plenum 400. A downwardly facing suction cup,such as 408 a-408 d, is attached at the lower end of each leg 406 a-406d. The face region of each suction cup 408 a-408 d is disposed in fluidcommunication with plenum 400 via a channel (not shown) extendinglongitudinally through a respective leg 406 a-406 d. Plenum 400, inturn, communicates with a remote vacuum source (not shown) via asuitable hose. Upon evacuating plenum 400, a vacuum is established atthe face region of each suction cup 408 a-408 d.

With additional reference to FIG. 24, platen 412 is a multi-layeredassembly, including (from top to bottom) (i) a support plate 414; (ii) aheat-insulating layer 416; (iii) a heater 418; and (iv) a thermallyconductive, conformable pad 420; each of which is described more fullybelow.

Support plate 414 is formed of a rigid material that, when pressed downfrom above, is capable of transmitting the downward pressure across thevarious underlayers 416, 418, 420, so that the lower surface of thethermally conductive, conformable pad 420 is pressed against the uppersurface of a multi-well tray. Suitable materials for forming supportplate 414 include metals, such as aluminum and the like. A plurality ofdepressions or indentations, such as 425-428, are provided along theupper surface of plate 414, providing landing sites, or seats, for thefingers of a fluid-handling robot, such as a four-fingered TECAN® RSP,to abut and press down upon the platen 412, as described below.

The heat-insulating layer 416 thermally isolates the upper support plate414, and associated elements, from heat and hot components thereunder.In one preferred embodiment, the heat-insulating material is a phenolicblock.

The heater 418 is preferably an electrically resistive heating element(not shown) disposed within a heat-conductive metallic plate, such as analuminum plate or the like. The heating element can be, for example, asilicone rubber heater. A preferred silicone rubber heater (80 Watts, 24Volts) is available commercially from Minco Products, Inc. (Minneapolis,Minn.).

The thermally conductive, conformable pad 420 acts as a thermalinterface between the heated metallic plate and the area along the uppersurface of a multi-well plate. A preferred material for forming the padis available commercially under the trade name Gap Pad™ from TheBergquist Company; (Edina, Minn.). Gap Pad™ is a highly conformablesilicone polymer filled with alumina (See, e.g., U.S. Pat. No.5,679,457; expressly incorporated herein by reference). The pad,attached to the underside of the heated metallic plate, has a thicknessof about 0.10″ to 0.20″, and preferably about 0.160″. The pad provides aheated surface capable of conforming to the contours of the uppersurface of the multi-well plate for applying a heat-sealable sheetthereto.

Platen 412 further includes a frame structure having two substantiallyvertical side panels 432, 434 held in fixed spaced relation by a pair ofrectangular crossbar members, such as crossbar 438. The crossbar membersare rigidly attached to the side panels 432, 434, e.g., by way offasteners 440, so as to form narrow, flat floor regions bridging theconfronting faces of the side panels 432, 434, at each longitudinal endthereof (only one of which is visible in the figures). In an alternativeembodiment, the frame structure is cast as a unitary piece.

Each end of support plate 414 has an overhang region, such as 414 a and414 b, that projects longitudinally beyond the various underlayers 416,418, 420. Each overhang region 414 a, 414 b is about the same size,along its length and width dimensions, as one of the crossbar members.As best seen in FIG. 24, the lower surface of overhang region 414 afaces the upper surface of a crossbar member 438. Although not visiblein the figures, it should be noted that the same arrangement exists onthe opposing side of the frame structure.

Three elongated, cylindrical rods are disposed substantially normal toan upper, flat surface of each crossbar member, at spaced pointsgenerally defining a line. For example, FIG. 24 shows rods 450, 452, 454near one end of platen 412. Similar structure exists near the other endof platen 412, as well. The lower end of each rod is rigidly attached toits respective crossbar member, while the upper (free) end is passedthrough a respective bore (not shown) formed vertically through arespective overhang region, 414 a or 414 b. The two outer rods on eachcrossbar member, such as rods 450 and 454, are received in linearbearings, such as bearings 460 and 464, held within such bores, andserve to guide the platen 412 as it moves up and down along a generallyvertical direction. The center rod on each crossbar member, such as rod452, forms a part of a biasing means that acts to urge the platen 412upward. In this regard, a compression spring, such as spring 468, isconcentrically mounted about the center rod at each end of platen 412,with the spring pre-compressed between the upper surface of itsrespective crossbar member and the lower surface of a confrontingoverhang region, 414 a or 414 b. In its desire to extend, the springprovides a continuous, upwardly-directed force that, in the absence ofan equal or greater opposing force, is sufficient to position platen 412in a fully raised position, whereat the support plate 414 is disposedproximate the top edge regions of side panels 432, 434.

An E-style retaining ring, such as 470 and 471, is mounted near the topof each center (spring-bearing) rod, limiting the upward movement of thesupport plate 414. In an alternative embodiment (not shown), the upperedge of each side panel can be angled inward, e.g., 90 degrees relativeto the major surface of the panel, to form a lip that acts to limit theupward movement of the support plate.

As previously indicated, pick-and-place assembly 388 is used to pick upindividual heat-sealable covers from a holding tray 336 and place themon a multi-well tray 324. The heatable platen 412 applies the heat andforce necessary for effecting a proper seal.

In an exemplary operation, and with reference to FIGS. 16-24, the heatsealing station apparatus begins the sealing process by rotating pickerarm 390 from the home position (FIGS. 16 and 23) to the pick-up position(FIGS. 17 and 20), through an arc of about 90°. Carriage 376 moves alonglinear track 370, as necessary, until suction picker assembly 394 ispositioned directly above a tray 336 holding a stack ofpolyethylene/polypropylene covers 342. Here, suction picker assembly 394is driven down, by way of stepper motor 402 and lead screw 404, untileach suction cup 408 a-408 d contacts the uppermost sheet of stack 342.Plenum 400 is then evacuated by a remote vacuum source (not shown) inorder to establish a vacuum, e.g., from about −5 to about −10 psi, atthe face region of each suction cup 408 a-408 d. Suction picker assembly394 is then driven up, thereby lifting a heat-sealable cover 342 a fromstack 342. Picker arm 390 is then rotated another 90°, from the pick-upposition to a drop-off position (FIGS. 21 and 22). Here, suction pickerassembly 394 is driven down until sheet 342 a rests on a multi-well tray324, at which point the vacuum is released. Suction picker assembly 394is then raised back up, while suction picker arm 390 is returned to thehome position. Next, carriage 376 is moved forward to position platen412 directly above multi-well tray 324. The TECAN® RSP 198 (FIG. 16) ismoved along the x/y direction to position its four fingers 1-4 aboverespective landing sites 425-428 on top of support plate 414. The TECAN®RSP then presses down, along the z direction, with each of fingers 1-4,thereby compressing the bottom surface of platen 412, heated to about105°-120° C., against the heat-sealable sheet 342 a resting onmulti-well tray 324. Heated platen 412 is held against the multi-welltray for a short period (e.g., about 10-20 seconds), at a pressure ofabout 20 lbs., thereby sealing the heat-sealable sheet 342 a onto tray324. The fingers 1-4 of the TECAN® RSP 198 are then raised, therebyallowing the heated platen 412 to raise. The above process is repeatedfor any other multi-well plates held in a cavity 310 of work surface302.

As previously mentioned, a computer control unit can be programmed,using known techniques, to automate the above process. To this end,non-contact sensors, for example infrared emitter/detector pairs (notshown), and sensor flags, such as flag 476, can be strategicallypositioned to provide position signals for monitoring by the computercontrol unit.

Those skilled in the art can now appreciate from the foregoingdescription that the broad teachings of the present invention can beimplemented in a variety of forms. Therefore, while this invention hasbeen described in connection with particular embodiments and examplesthereof, the true scope of the invention should not be so limited.Various changes and modification may be made without departing from thescope of the invention, as defined by the appended claims.

It is claimed:
 1. An apparatus for avoiding cross-contamination due topendent drops of fluid hanging from a plurality of discharge conduitsdisposed in an array above a corresponding array of receiving wells,comprising: (i) a carriage configured to carry one of said arrays andadapted for linear reciprocal motion in either of two directions along afirst, generally horizontal, axis from a neutral position whereat saidarrays are substantially axially aligned; (ii) a stepper motor; (iii) alinkage assembly mechanically communicating said stepper motor with saidcarriage such that each rotational step of said stepper motor inducesmovement of said carriage a given distance from said neutral position inone of said two directions depending upon the direction of angularrotation of said motor, thereby effecting relative motion between saiddischarge-conduit array and said receiving-well array such that pendentdrops of fluid hanging from said discharge conduits are simultaneouslytouched-off to inner sidewalls of corresponding receiving wells; and(iv) a compression spring mounted within said linkage assembly in amanner permitting said spring (a) to provide a predetermined amount ofresistance to movement of the carriage from said neutral position, and(b) to compensate or absorb linear overshoot due to excess angularrotation of the motor beyond the amount required to move the dischargeconduits into firm abutment with the inner sidewalls of the receivingwells.
 2. An apparatus for avoiding cross-contamination as recited inclaim 1 further comprising: a vacuum chamber communicating with saiddischarge-conduit array from a side thereof opposite said receiving-wellarray, whereby evacuation of said vacuum chamber is effective to urgependent drops of fluid hanging from said discharge conduits in adirection away from said receiving wells and into said dischargeconduits.
 3. An apparatus for avoiding cross-contamination as recited inclaim 1, wherein said carriage is configured to carry saiddischarge-conduit array.
 4. An apparatus for avoidingcross-contamination as recited in claim 3, further comprising: avertical positioning assembly disposed on said carriage and supportingsaid discharge-conduit array for linear movement along a second,generally vertical, axis between a lowered position whereat saiddischarge conduits extend down into respective receiving wells and anelevated position whereat said discharge conduits clear said receivingwells.
 5. A method for avoiding cross-contamination due to pendent dropsof fluid hanging from a plurality of discharge conduits disposed in anarray above a corresponding array of receiving wells, comprising: (i)touching-off, in a substantially simultaneous fashion, said pendentdrops of fluid hanging from said discharge conduits to inner sidewallsof respective receiving wells; and (ii) drawing said pendent drops offluid hanging from said discharge conduits in a direction away from saidcorresponding receiving-well array and into said discharge conduits. 6.The method of claim 5, wherein said step of touching-off is effected byshifting the discharge-conduit array along a plane substantiallyorthogonal to the longitudinal axes of the receiving wells, while thereceiving wells are maintained in a substantially fixed position.
 7. Themethod of claim 6, wherein each of said discharge conduits is shiftedinto contact with one sidewall portion of a respective receiving well,and then is shifted into contact with another, laterally opposingsidewall portion of the same receiving well.
 8. The method of claim 7,wherein a stepper motor is stepped in order to shift saiddischarge-conduit array, said stepper motor disposed in mechanicalcommunication with said discharge-conduit array such that angularrotation of said stepper motor induces linear motion of said dischargeconduits.
 9. The method of claim 5, wherein said step of drawing pendentdrops of fluid is effected by establishing a reduced pressure above saiddischarge conduits.
 10. The method of claim 5, wherein an upstandingupper end region of each of said discharge conduits is received within arespective column, thereby forming an array of microfiltration wells;and wherein each column has (i) a first inner bore defining a lumenwithin the column, and (ii) an end region defining (a) a second innerbore having a diameter greater than that of the first inner bore and (b)a transition region that joins the second inner bore to the first innerbore; and wherein a filter element is disposed in each column, saidfilter element held in compression between the transition region of thecolumn and the upper end region of a respective discharge conduit.